WO2025189158A1 - Vaccins à matrice de micro-aiguilles à adjuvant de polyphosphazène - Google Patents

Vaccins à matrice de micro-aiguilles à adjuvant de polyphosphazène

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Publication number
WO2025189158A1
WO2025189158A1 PCT/US2025/019036 US2025019036W WO2025189158A1 WO 2025189158 A1 WO2025189158 A1 WO 2025189158A1 US 2025019036 W US2025019036 W US 2025019036W WO 2025189158 A1 WO2025189158 A1 WO 2025189158A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
antigen
microneedle array
pathogen
dissolvable microneedle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/019036
Other languages
English (en)
Other versions
WO2025189158A8 (fr
Inventor
Emrullah Korkmaz
Jr. Louis D. Falo
Christopher Broder
Alexander Andrianov
Antony DIMITROV
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Pittsburgh
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
University of Maryland College Park
Original Assignee
University of Pittsburgh
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
University of Maryland College Park
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Pittsburgh, Henry M Jackson Foundation for Advancedment of Military Medicine Inc, University of Maryland College Park filed Critical University of Pittsburgh
Publication of WO2025189158A1 publication Critical patent/WO2025189158A1/fr
Publication of WO2025189158A8 publication Critical patent/WO2025189158A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
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    • A61K9/0012—Galenical forms characterised by the site of application
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    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays or needleless injectors
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Definitions

  • FIELD This relates to the field of vaccines, specifically to a polyphosphazene adjuvanted microneedle array and its use to generate an immune response.
  • INCORPORATION OF ELECTRONIC SEQUENCE LISTING The electronic sequence listing, submitted herewith as an XML file named 8123-111083- 02_Sequence Listing.xml (26,509 bytes), created on February 20, 2025, is herein incorporated by reference in its entirety.
  • BACKGROUND The threat of (re)-emerging infectious pathogens highlights the need for continued improvement of immunization strategies through the development of more efficacious, safer, and more broadly deployable vaccines than existing vaccines to enable sustainable and equitable global immunization campaigns.
  • Vaccines can be administered through various routes of delivery, including the oral, nasal, intramuscular (IM) or intradermal (ID) route.
  • routes of delivery including the oral, nasal, intramuscular (IM) or intradermal (ID) route.
  • IM intramuscular
  • ID intradermal
  • Vaccines can be administered through various routes of delivery, including the oral, nasal, intramuscular (IM) or intradermal (ID) route.
  • IM intramuscular
  • ID intradermal
  • Vaccines can be administered through various routes of delivery, including the oral, nasal, intramuscular (IM) or intradermal (ID) route.
  • IM intramuscular
  • ID intradermal
  • Microneedle arrays represent an ideal needle-free, self-administered, and thermostable vaccine delivery platform that can precisely and reproducibly target vaccine components to the immunoresponsive skin microenvironments for efficacious immunization.
  • a need remains for new adjuvants that can be effectively used to boost immunity to various immunogens when used in microneedle array format.
  • SUMMARY Provided herein are broadly applicable polyphosphazene adjuvanted microneedle array based skin-targeted vaccine platforms that are capable of eliciting broadly protective immunity against target pathogen(s) or tumor(s).
  • Microneedle array is used interchangeably with microneedle patch herein.
  • a dissolvable microneedle array that includes a base portion (such as a flat rigid, or flat conformable base portion) and a first plurality of microneedles extending from the base portion, each microneedle of the first plurality including a polyphosphazene adjuvant and a first antigen from a first pathogen (or tumor), wherein the first plurality of microneedles includes an amount of the polyphosphazene adjuvant and the first antigen effective to elicit an immune response to the first antigen or first pathogen (or tumor) in a subject.
  • the polyphosphazene adjuvant is poly[di(carboxylatoethylphenoxy)phosphazene] (PCEP), fluorinated PCEP, PEGylated PCEP, poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene], or any combination thereof; and the first pathogen is a virus or bacterium.
  • the polyphosphazene adjuvant is poly[di(carboxylatophenoxy)phosphazene] (PCPP), fluorinated PCPP, PEGylated PCPP, poly[ di(carboxylatophenoxy)(trifluoroethoxy)phosphazene], or any combination thereof; and the first pathogen is a virus or a bacterium.
  • PCPP poly[di(carboxylatophenoxy)phosphazene]
  • fluorinated PCPP PEGylated PCPP
  • poly[ di(carboxylatophenoxy)(trifluoroethoxy)phosphazene] or any combination thereof
  • the first pathogen is a virus or a bacterium.
  • disclosed is the use of these polyphosphazene adjuvanted microneedle array vaccines to induce an immune response to the pathogen. 8123-111083-02
  • FIG.1 Schematic diagrams of various exemplary microneedle geometries.
  • FIG.1 is identical to FIG.20 of U.S. Published Patent Application US-2019-0000966-A1, which is incorporated herein by reference.
  • FIG.2 Illustrations of exemplary microneedle geometries that have sharp-tipped conical heads, circular undercut stems, and filleted bases.
  • FIG.2 is identical to FIG.1 of U.S. Published Patent Application US-2022-0241570-A1, which is incorporated herein by reference.
  • FIG.3 A scanning electron microscope (SEM) image of a micromilled mastermold with pyramidal needles.
  • FIG.3 is identical to FIG.8 of U.S.
  • FIGS.4A and 4B An SEM image of a pyramidal production mold (FIG.4A), and an SEM image of an enlarged segment of the production mold, illustrating a pyramidal needle molding well in the center of the image (FIG.4B).
  • FIGS.4A and 4B are identical to FIGS.9 and 10, respectively, of U.S. Published Patent Application US-2016-0271381-A1, which is incorporated herein by reference.
  • FIG.5 Schematic diagrams of the different steps of an exemplary additive manufacturing method for microneedle array production.
  • FIG.5 is identical to FIG.4B of U.S.
  • FIG.6 Schematic diagrams of an exemplary multi-layered CMC solid with active cargos deposited therein and methods of creating the same, and a microneedle array (MNA) micro-milled from the CMC solid.
  • MNA microneedle array
  • FIG 6 is identical to FIG.13 of U.S. Published Patent Application US-2016-0271381-A1, which is incorporated herein by reference.
  • FIG.7 Schematic diagrams of polyphosphazene-complexed antigens, and a microneedle array loaded with the polyphosphazene-complexed antigens.
  • FIG.8D is a microscopy image of an MNP loaded with HeV-sG and poly[di(carboxylatoethylphenoxy) phosphazene] (PCEP).
  • FIGS.9A-9B Images showing in vivo skin penetration and dissolution performance of polyphosphazene adjuvanted MNP vaccines.
  • FIG.9A shows microscopy images of MNPs loaded with HeV-sG labeled with AF555 dye (HeV-sG AF555), or with HeV-sG AF555 and PCEP, before and after application.
  • HeV-sG AF555 AF555 dye
  • FIG.9B shows in vivo live animal fluorescent analysis images of mice administered with MNPs loaded with HeV-sG AF555, or with MNPs loaded with HeV-sG AF555 and PCEP, demonstrating that polyphosphazene adjuvanted MNP vaccines effectively penetrate the skin, dissolve, and deliver vaccine components.
  • FIGS.10A-10B Bar graphs showing the in vivo immunogenicity of polyphosphazene adjuvanted MNP vaccines.
  • FIG.10A shows anti-HeV-sG total IgG endpoint titers (EPTs) at week 2 and week 5.
  • FIG. 10A shows anti-HeV-sG total IgG endpoint titers (EPTs) at week 2 and week 5.
  • FIGS.11A-11B Bar graphs showing the in vivo immunogenicity of polyphosphazene adjuvanted MNP vaccines.
  • Four groups of C57BL/6 mice (n 5) were used: one group was not immunized (na ⁇ ve); one group was immunized with a MNP loaded with HeV-sG; one group was immunized with MNPs loaded with HeV-sG and PCPP; one group was immunized with MNPs loaded with HeV-sG and PCEP.
  • the treated groups were administered with a primer dose at week 0, and with a booster dose at week 3.
  • FIG.11A shows anti-HeV-sG total IgG EPTs at week 5, week 8, and week 16.
  • FIG.11B shows virus neutralization titers (NT50) at week 5, week 8, and week 16.
  • FIG.12 Bar graph showing the in vivo immunogenicity of polyphosphazene adjuvanted MNP vaccines.
  • the treated groups were 8123-111083-02 administered with a primer dose at week 0, and with a booster dose at week 3. Blood samples were taken from all groups at week 2, week 5, week 8, week 16, week 24, week 32, and week 40, and sera were assayed by ELISA.
  • FIG.6 shows anti-HeV-sG total IgG EPTs at week 2, week 5, week 8, week 16, week 24, week 32, and week 40.
  • FIG.13 Bar graph illustrating the dose-sparing of polyphosphazene adjuvanted MNP vaccines.
  • the treated groups were immunized at week 0. Blood samples were taken from all groups at week 2, and sera were assayed by ELISA.
  • FIG.13 shows anti-HeV-sG total IgG EPTs at week 2.
  • FIG.14 Bar graph showing the temperature stability of polyphosphazene adjuvanted MNP vaccines.
  • FIG.14 shows anti-HeV-sG total IgG EPTs at week 2.
  • FIG.15A shows the amount of antigen-specific CD4 + cells per spleen.
  • FIG.15B shows the amount of antigen- specific CD8 + cells per spleen.
  • FIG.16 Kaplan-Meier survival plot showing the in vivo protection efficacy of polyphosphazene adjuvanted MNP vaccines.
  • FIGS.17A-17D Broad application and superior immunogenicity of polyphosphazene adjuvanted MNP vaccines against several pathogens, including SARS-CoV- 2 virus, influenza virus, Sudan ebolavirus, and Hepatitis C virus. Mice were immunized by polyphosphazene adjuvanted MNP vaccines and/or traditional needle-and-syringe vaccines through intramuscular injection. Serum levels of antigen-specific total IgG antibody were measured by ELISA.
  • FIG.17A Bar graphs showing higher serum concentrations of SARS-CoV-2 spike protein (SP)-specific total IgG in mice immunized by polyphosphazene adjuvanted MNP- based SARS-CoV-2 SP vaccine, compared to those in mice vaccinated by intramuscular injection of conventional aluminum adjuvanted SARS-CoV-2 SP vaccine.
  • FIG.17B Bar graphs showing higher serum concentrations of influenza hemagglutinin (HA)-specific total IgG in mice immunized by polyphosphazene adjuvanted MNP-based influenza HA vaccine, compared to those in mice immunized by unadjuvanted MNP-based influenza HA vaccine.
  • SP SARS-CoV-2 spike protein
  • FIG.17C Line graphs showing higher serum levels of Sudan ebolavirus glycoprotein (GP)-specific total IgG in mice immunized by polyphosphazene adjuvanted MNP-based Sudan ebolavirus GP vaccine, compared to those in mice immunized by intramuscular injection of conventional aluminum adjuvanted Sudan ebolavirus GP vaccine.
  • GP Sudan ebolavirus glycoprotein
  • FIG.17D Line graphs showing higher serum levels of Hepatitis C virus sE1E2- specific total IgG in mice immunized by polyphosphazene adjuvanted MNP-based Hepatitis C virus sE1E2 vaccine, compared to those in mice immunized by intramuscular injection of conventional aluminum adjuvanted Hepatitis C virus sE1E2 vaccine.
  • FIGS.18A-18C Bar graphs showing the in vivo immunogenicity of polyphosphazene and/or poly(I:C) adjuvanted MNP vaccines.
  • FIG.18A shows antigen-specific antibody responses.
  • the treated groups were administered with a primer dose at week 0. Blood samples were taken from all groups at week 2, and sera were assayed by ELISA.
  • FIG.18A shows anti-HeV-sG total IgG EPTs at week 2.
  • FIGS.18B and 18C show antigen-specific cellular immune responses.
  • Five groups of C57BL/6 mice (n 5) were used: one group was not immunized (na ⁇ ve); one group was immunized with a MNP loaded with HeV-sG; one group was immunized with a MNP loaded with HeV-sG and poly(I:C); one group was immunized with a MNP loaded with HeV-sG and PCEP; one group was immunized with HeV-sG and poly(I:C) and PCEP.
  • the immunized groups were administered with a primer dose at week 0 and a booster dose at week 3. All groups were sacrificed 5 days after the last dose.
  • FIG.18B shows the amount of antigen-specific CD4 + cells per spleen.
  • FIG. 18C shows the amount of antigen-specific CD8 + cells per spleen.
  • FIGS.19A-19E PCEP adjuvanted MNP-based henipavirus vaccine induces robust and long-lasting virus-specific (homologous) and cross-reactive (heterologous) humoral responses in a prime-boost regimen in mice.
  • FIG.19A is a schematic that summarizes the experimental process.
  • Serum levels of binding and neutralizing antibodies were measured in mice that received two doses of HeV- sG + PCEP MNP or HeV-sG MNP vaccine three weeks apart by the application of MNPs for 15 min to their abdomen for primer and booster doses, respectively. Naive mice served as unimmunized controls. Both homologous (HeV-specific) and heterologous (NiV-specific) total IgG binding antibody endpoint titers and neutralizing antibody levels were measured 5, 8, 16, 52, and 72 weeks following the primer vaccination by ELISA and in vitro chimeric cedar virus-based surrogate neutralization assays, respectively.
  • FIGS.19B and 19C are plots that show homologous humoral responses elicited by HeV-sG + PCEP MNP or HeV-sG MNP vaccine, where FIG.19B shows anti-HeV-G total IgG endpoint titers, and FIG.19C shows the 50% neutralization dose (titers) (ND50) against HeV.
  • FIGS.19D and 19E are plots that show heterologous humoral responses elicited by HeV-sG + PCEP MNP or HeV-sG MNP vaccine, where FIG.19D shows anti-NiV-G total IgG endpoint titers, and FIG.19E shows the 50% neutralization dose (titers) (ND 50 ) against NiV.
  • FIGS.20A-20D PCEP adjuvanted MNP-based henipavirus vaccine induces robust and long-lasting virus-specific (homologous) and cross-reactive (heterologous) humoral responses in a single-dose regimen in mice.
  • FIG.20A is a schematic that summarizes the experimental process. Serum levels of binding antibodies were measured in mice that received a single dose of HeV-sG + PCEP MNP or HeV-sG MNP vaccine by the application of MNPs for 15 min to their abdomen. Naive mice served as unimmunized controls.
  • FIG.20B is a plot that shows anti-HeV-G total IgG endpoint titers elicited by HeV-sG + PCEP MNP or HeV-sG MNP vaccine.
  • FIG.20C is a plot that shows anti-NiV-G total IgG endpoint titers elicited by HeV-sG + PCEP MNP or HeV-sG MNP vaccine.
  • FIG.20D shows correlation between anti-HeV-G total IgG endpoint titers and anti- NiV-G total IgG endpoint titers.
  • FIGS.21A-21C PCEP adjuvanted MNP-based henipavirus vaccine induces significantly improved Th1-skewing cellular responses compared to benchmark groups in a prime-boost regimen in mice.
  • FIG.21A is a schematic that summarizes the experimental process. Mice received two doses of HeV-sG + PCEP MNP, HeV-sG + PCPP MNP, or HeV-sG +Alum vaccine three weeks apart, where the MNP vaccines were applied for 15 min to the abdomen of mice, and the HeV-sG + Alum vaccine was administered intramuscularly.
  • FIG.22B is a Kaplan-Meier survival plot that shows percentage of ferrets survived in each group up till 28 days after infection, demonstrating the in vivo protection efficacy of HeV-sG + PCEP MNP.
  • FIG.22C is a line graph that shows average clinical score for ferrets in each group up till 28 days after infection.
  • FIG.22D is a bar graph that shows viremia for ferrets in each group up till 28 days after infection.
  • FIG.22E is a bar graph that shows concentration of virus found in spleen, kidney, and lung for ferrets in each group at day 49 after infection.
  • FIGS.23A-23B PCEP adjuvanted MNPs immunomodulates the human skin microenvironment to induce potent antigen-presenting cells with immunostimulatory phenotypes. MNPs were applied to human skin explants. After 5 minutes, MNPs were removed, and explants were placed with epidermis up on steel mesh rafts with AIM-V serum- free media supplemented with 2.5 mg/mL Amphotericin B (HyClone), and cultured for 48 hours at 37 °C in 5% CO2.
  • AIM-V serum- free media supplemented with 2.5 mg/mL Amphotericin B (HyClone)
  • FIG.23A Contour plots that show the subsets of skin migratory dendritic cells 48 hours after HeV-sG + PCEP MNP or HeV-sG MNP treatment.
  • FIG.23B Histograms showing the mean fluorescent intensity for markers HLA-DR, CD40, CD80, CD83, CD86, or CCR7, 48 hours after HeV-sG + PCEP MNP or HeV-sG MNP treatment.
  • FIGS.24A-24D PCEP adjuvanted MNPs achieve safe and effective vaccination without signs of vaccine reactogenicity.
  • FIG.22A is a schematic that summarizes the experimental process. Mice received a single dose of HeV-sG + PCEP MNP vaccine at day 0. Serum samples from the mice were collected before vaccination, and at day 1, which were then measured for IL-6 cytokine levels by ELISA. Body weight and temperature of the mice were measured before vaccination, and at days 1, 4, and 7. 8123-111083-02
  • FIGS.22B and 22C are bar graphs that show average body weight and body temperature of mice before vaccination, and at days 1, 4, and 7.
  • FIG.22D shows that serum IL-6 levels of mice are below the limit of quantification both before vaccination and at day 1.
  • FIGS.25A-25B PCEP adjuvanted MNPs are thermostable.
  • FIG.25A is a schematic that summarizes the experimental process. Mice received, at week 0, a single dose of 1) freshly prepared HeV-sG + PCEP MNP vaccine, 2) HeV-sG + PCEP MNP vaccine stored at room temperature for 1 year, 3) ⁇ -irradiated HeV-sG + PCEP MNP vaccine stored at room temperature for 1 year, or 4) HeV-sG + PCEP MNP vaccine stored at 40 °C for 3 months. Serum samples from the mice were collected at week 4, which were measured for anti-HeV-G total IgG levels by ELISA.
  • FIG.25B is a bar graph that shows anti-HeV-G total IgG endpoint titers for mice receiving 1), 2), 3) or 4).
  • DETAILED DESCRIPTION The present disclosure provides a polyphosphazene adjuvanted dissolvable microneedle patch vaccine platform (a microneedle array) that is readily adaptable with different vaccine constructs to develop efficacious, broadly deployable vaccines against emerging and re-emerging pathogens.
  • the disclosed polyphosphazene adjuvanted microneedle patch vaccine platform is thermostable, and thus can be stored and transported easily without the need for refrigeration.
  • microneedle patch vaccines are skin-targeted vaccines that can efficiently harness the highly immunoresponsive skin microenvironments, thereby generating long-lasting systemic and mucosal immune responses.
  • the disclosed polyphosphazene adjuvanted dissolvable microneedle patch vaccine can elicit robust, strong, and durable protective immunity against the target pathogens.
  • a polyphosphazene adjuvanted dissolvable microneedle patch vaccines can be self-administered.
  • the disclosed microneedle arrays that include a pathogen-specific antigen and a polyphosphazene adjuvant can be used to generate a highly potentiated protective immune response against a pathogen.
  • the immune response can provide cross- protection against a related pathogen.
  • the polyphosphazene adjuvant is PCEP, fluorinated PCEP, PEGylated PCEP, poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene], or any combination thereof; and the first pathogen is a virus or bacterium.
  • the 8123-111083-02 polyphosphazene adjuvant is PCPP, fluorinated PCPP, PEGylated PCPP, poly[di(carboxylatophenoxy)(trifluoroethoxy)phosphazene], or any combination thereof and the first pathogen is a bacterium or virus.
  • the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain aspects of the disclosure are to be understood as being modified in some instances by the term “about” or “approximately.” Accordingly, in some aspects, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained by a particular aspect. In some aspects, the term “about” or “approximately” indicates within five percent. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
  • Adjuvant A substance or vehicle that enhances an immune response to an antigen, or when combined with an antigen elicits an immune response to the antigen.
  • Polyphosphazene (such as those disclosed herein) can function as an adjuvant.
  • adjuvants include, but are not limited to, Freund’s adjuvant, toll-like receptor (TLR) agonist (such as resiquimod, imiquimod, a CpG oligodeoxynucleotide, or poly(I:C)), a cGAS/STING pathway agonist (such as ADU-S100, or MK-1454), or a saponin adjuvant (such as QS-21).
  • TLR toll-like receptor
  • TLR toll-like receptor
  • cGAS/STING pathway agonist such as ADU-S100, or MK-1454
  • saponin adjuvant such as QS-21.
  • Antibody An immunoglobulin molecule produced by B lymphoid cells of humans or other animals upon activation by an antigen, or an antigen in combination with an adjuvant. An antibody specifically recognizes and interacts with an antigen, and can therefore be characterized or defined by the antigen it recognizes and interacts with.
  • Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • the term “antigen” includes all related antigenic epitopes. “Epitope” or “antigenic determinant” refers to a site on an antigen, such as a polypeptide antigen, to which B and/or T cells respond. In one aspect, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, at least 5, at least 9, at least 10, at least 11, at least 12, or about 9-12 amino acids in a unique spatial conformation.
  • Antigens may be proteins (including peptides and polypeptides), polysaccharides, and lipids.
  • Antigens may be purified molecules (as in subunit vaccines), linked to a carrier, or part of or carried by an inactivated pathogen, attenuated pathogen, or pathogen-like particle. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
  • Active component Used interchangeably with active cargo or active ingredient. A component that exerts the intended effect upon administration to a subject, including antigens, nucleic acids encoding antigens, inactivated pathogens, attenuated pathogens, pathogen-like particles, and adjuvants.
  • Attenuated pathogen Used interchangeably with live-attenuated pathogen.
  • a pathogen e.g., virus, bacterium, fungus, or protozoan
  • a pathogen is attenuated if its ability to infect a 8123-111083-02 cell or subject and/or its ability to produce disease is reduced or eliminated compared to a wild-type pathogen.
  • an attenuated pathogen retains at least some capacity to elicit an immune response following administration to an immunocompetent or an immunocompromised subject.
  • an attenuated pathogen is capable of eliciting a protective immune response without causing any signs or symptoms of infection.
  • an attenuated pathogen to cause disease in a subject is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90% relative to a wild-type pathogen.
  • An attenuating mutation is a mutation in a pathogen genome that results in an attenuated pathogen.
  • Base material Material used to form a microneedle array other than active components. A base material provides structure and shape to a microneedle array. Base materials used to form the portions of a microneedle array that enter the skin are dissolvable and biocompatible, while base materials used to form the portions of a microneedle array that do not enter the skin can be dissolvable or not.
  • Biocompatible The property of not causing undesirable local or systemic effects in a subject when administered at an amount effective for its purpose.
  • a biocompatible material e.g., polymer
  • biocompatible materials include, without limitation, carboxymethylcellulose (CMC), trehalose, sucrose, gelatin, glucose, lactose, collagen, chitosan, poly- ⁇ -glutamate, polyvinylpyrrolidone (PVP), maltodextrin, silk, hyaluronic acid (HA), methacrylated hyaluronic acid (MeHA), poly(lactic-co-glycolic acid) (PLGA), poly(vinylalcohol) (PVA), polyethylene glycol (PEG), PEG-crosslinked poly(methyl vinyl ether-co-maleic acid), or any combination thereof.
  • CMC carboxymethylcellulose
  • trehalose sucrose
  • gelatin glucose
  • lactose collagen
  • collagen chitosan
  • poly- ⁇ -glutamate polyvinylpyrrolidone
  • PVP polyvinylpyrrolidone
  • MeHA methacrylated hyaluronic acid
  • PLGA poly(lactic-co-gly
  • Biological samples include, for example, fluid, cell and/or tissue samples.
  • Fluid samples include, but are not limited to, serum, blood, plasma, urine, feces, saliva, sputum, semen, cerebral spinal fluid (CSF).
  • Tissue samples can be from any organ (such as skin, lung, spleen, liver, kidney, heart, brain, etc.) and obtained through biopsy procedures, or obtained during surgical procedures.
  • Carrier A molecule or particle which an antigen can be linked to or associated with. When linked to or associated with a carrier, the antigen may become more immunogenic. Carriers are chosen to increase the immunogenicity of the antigen and/or to elicit antibodies against the carrier which are diagnostically, analytically, and/or therapeutically beneficial.
  • Useful carriers include polymeric carriers (which can be natural (for example, proteins from 8123-111083-02 bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached), and nanoparticle carriers, which enhance immunogenicity of an antigen by more closely resemble a pathogen.
  • cGAS/STING pathway agonist A substance that activates cGAS/STING pathway.
  • cGAS/STING pathway is part of the innate immune system that detects cytosolic DNA and, in response, trigger expression of inflammatory genes that can lead to senescence or to the activation of defense mechanisms.
  • Conservative substitution A substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties.
  • conservative substitutions have little to no impact on the activity of a resulting polypeptide.
  • a Hendra virus G glycoprotein variant including one or more conservative substitutions for example no less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 substitutions, for example 2 to 50, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 5 substitutions
  • a polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Examples of conservative substitutions are shown below.
  • substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted by a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted by any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted by an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted by one not having a side chain, for example, glycine.
  • a hydrophilic residue for example, seryl or threonyl
  • a hydrophobic residue for example, leucyl, is
  • Contacting Placing two substances (e.g., molecules, cells, liquids, solids, gases, etc., or any combination thereof), or a substance and a subject in physical touching, adjacency, or interaction. “Contacting” is often used interchangeably with “exposing.”
  • Control A reference standard, including a positive control and a negative control.
  • a positive control is known to provide a positive test result.
  • a negative control is known to provide a negative test result.
  • a control or reference standard can be a theoretical or computed result, for example a result obtained in a population.
  • Dissolvable Water-soluble or dissolvable in the skin.
  • a dissolvable microneedle array refers to a microneedle array wherein at least the portions of the microneedles that will be inserted into the skin upon use are dissolvable.
  • Effective amount A quantity of one or more active components that, when administered to a subject is sufficient to achieve a desired effect (such as eliciting an immune response, enhancing an immune response, preventing an infection or disease, treating a tumor, etc.). The quantity can be the amount in a plurality of microneedles that induces an immune response when the plurality of microneedles is administered to the subject.
  • An effective 8123-111083-02 amount will be dependent on, for example, the subject being treated, the route of administration, the substance(s) being administered, the desired result, and the like.
  • Fusion protein A protein generated by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain no internal stop codons.
  • F glycoprotein Used interchangeably with F protein, also known as the fusion protein or fusion glycoprotein. F glycoprotein refers to a protein found on the surface of viruses that mediates fusion of the viral envelope with a host cell membrane.
  • F glycoprotein has been found in many viruses, including respiratory syncytial virus (RSV), measles virus, human parainfluenza viruses, henipavirus (e.g., Nipah virus, Hendra virus), Newcastle disease virus, mumps virus, canine distemper virus, etc.
  • RSV respiratory syncytial virus
  • measles virus e.g., measles virus
  • human parainfluenza viruses henipavirus (e.g., Nipah virus, Hendra virus), Newcastle disease virus, mumps virus, canine distemper virus, etc.
  • a soluble F glycoprotein refers to a F glycoprotein that lacks a transmembrane anchor/domain, and in some examples, lacks a cytoplasmic tail. In some examples, a soluble F glycoprotein is secreted when recombinantly produced.
  • Glycoprotein Proteins that have carbohydrate groups attached to the polypeptide chain.
  • G glycoprotein Used interchangeably with
  • G glycoprotein refers to a protein found on the surface of viruses that mediates attachment of the virus to receptors on a host cell membrane (also known as the attachment protein or attachment glycoprotein), or refers to a protein found on the surface of viruses that mediates both attachment of the virus to host cell membrane receptors and fusion of the viral envelope with a host cell membrane.
  • G glycoprotein has been found in many viruses, including respiratory syncytial virus (RSV), henipavirus (e.g., Nipah virus, Hendra virus), chandipura virus, etc.
  • RSV respiratory syncytial virus
  • henipavirus e.g., Nipah virus, Hendra virus
  • chandipura virus etc.
  • a soluble G glycoprotein refers to a G glycoprotein that lacks a transmembrane anchor/domain, and in some examples, lacks a cytoplasmic tail.
  • a soluble G glycoprotein is secreted when recombinantly produced.
  • Heterologous Originating from a different genetic source or species.
  • Henipavirus (HNV) A genus of negative-strand RNA viruses in the family Paramyxoviridae. The genus includes the following species: Cedar henipavirus, Ghanaian bat henipavirus, Hendra henipavirus (HeV), Mojiang henipavirus, Nipah henipavirus ((NiV), and Langya Henipavirus. 8123-111083-02 Hendra henipavirus: Used interchangeably with Hendra virus or HeV.
  • Inactivated Pathogen A pathogen (e.g., virus, bacterium, fungus, or protozoan) is inactivated if it is killed or rendered incapable of reproduction. Common methods for inactivating pathogens in vaccine production include chemical inactivation (such as treatment with formaldehyde or beta-propiolactone), heat inactivation, and radiation inactivation.
  • Immune response A response of a cell in a subject’s immune system to an antigen.
  • Immune responses include adaptive immune responses (including humoral and cellular immune responses, such as B-cells producing antibodies, a T-cell response, etc.), and innate immune responses (including macrophages and neutrophils targeting pathogens for phagocytosis, a complement response, inflammation, etc.).
  • Isolated or purified An “isolated” or “purified” biological substance (such as a nucleic acid, peptide, protein, protein complex, or particle) is a biological substance that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components (e.g., other DNA, RNA, or proteins) in the organism in which the substance occurs.
  • An isolated or purified biological substance can be obtained through isolation or purification from samples obtained from an organism; recombinant expression or production in host cells followed by purification, and chemical synthesis followed by purification.
  • isolated does not require absolute purity; rather, it is intended as a relative term, for example, referring to being more enriched with (or having a higher concentration of) the substance, compared to a crude preparation or a natural environment from which the substance is purified.
  • a biological substance is purified if the substance represents at least 50%, at least 60%, at least 8123-111083-02 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or greater, of the total moles of non-solvent substances in a preparation.
  • Microneedle array Used interchangeably with microneedle patch.
  • a construct comprising a base portion and a plurality of microneedles extending from the base portion.
  • a microneedle is a micron-scale protrusion from the base portion, for example, no greater than 2000 ⁇ m in length and having a largest cross section no greater than 100,000 ⁇ m 2 .
  • the base portion is a structure underlying and interconnecting the microneedles to form an integral array or patch.
  • Nipah henipavirus Used interchangeably with Nipah virus or NiV.
  • Nucleic acid molecule A polymer formed by two or more nucleotides, including RNA and DNA, in any form (e.g., linear, circular, single stranded, double-stranded, hairpin, loop, etc.).
  • a nucleotide includes a ribonucleotide, deoxyribonucleotide or a modified form of either.
  • the term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.”
  • a nucleic acid molecule is usually at least 10 nucleotides in length, unless otherwise specified.
  • Nucleic acid encoding an antigen A nucleic acid, such as a RNA (such as a mRNA), that provides instruction for and results in production of a desired protein antigen in a subject upon administration.
  • Nanoparticle-formulated antigen Antigen formulated together with a nanoparticle, which is any particulate material with a size of 1 to 1000 nm, nontoxic to a subject, and suitable for antigen formulation. See, e.g., L. Zhao et al., Nanoparticle vaccines, Vaccine, 32 (2014), pp.327-337, https://doi.org/10.1016/j.vaccine.2013.11.069).
  • Nanoparticle- formulated antigens include any combinative form of antigens and nanoparticles, such as antigens attached to nanoparticles (such as by covalent conjugation), antigens encapsulated within nanoparticles (with or without attachment), antigens adsorbed on the surface of nanoparticles, or antigens mixed with nanoparticles.
  • suitable nanoparticles include polymeric nanoparticles, inorganic nanoparticles, liposomes, immunostimulating complex (ISCOM), and self-assembled proteins, and emulsions.
  • Operably linked A first nucleic acid is operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Operably linked nucleic acids include a 8123-111083-02 first nucleic acid contiguous with the 5 ⁇ or 3 ⁇ end of a second nucleic acid.
  • a second nucleic acid is operably linked to a first nucleic acid when it is embedded within the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid.
  • Pathogen An organism that infects a subject, causing a disease or pathological condition.
  • a pathogen may be, without limitation, a virus, bacterium, fungus, protozoan, helminth, ectoparasite, etc.
  • Pathogens include pathogenic parasites.
  • Pathogen-like particle Particles carrying an antigen, intended to mimic some properties of a pathogen in its interaction with the immune system but devoid of the genetic material required for replication.
  • Pathogen-like particle includes, without limitation, virus- like particle, outer membrane vesicle, and nanoparticle-formulated antigens.
  • Pharmaceutically acceptable carrier The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Poly (I:C 12U) refers to a structure formed when uridine is introduced into a poly(I:C) strand.
  • Polypeptide A polymer formed by two or more amino acids. Polypeptide is used interchangeably with peptide and protein. 8123-111083-02
  • Polyphosphazene A polymer comprising alternating phosphorus and nitrogen atoms as the backbone, and side groups covalently bonded to the phosphorous atoms.
  • a polyethylene glycol (PEG)ylated polyphosphazene refers to a polyphosphazene wherein at least one side group is a PEG chain.
  • a halogenated (e.g., fluorinated, brominated, chlorinated, or iodinated) polyphosphazene refers to a polyphosphazene wherein at least one side group includes a halo (e.g., -F, -Cl, -Br, or -I), and/or haloalkyl (e.g., -CF3, -CCl3, -CBr3, or -CI 3 ) substituent.
  • Preventing a disease or infection refers to inhibiting the occurrence or full development of a disease or infection, such as a bacterium or virus infection, upon exposure to a disease causing substance or a pathogen.
  • “Ameliorating” refers to reducing the number or severity of one or more signs or symptoms of a disease or infection.
  • “Treating” refers to a therapeutic intervention that ameliorates or fully cures a disease or infection.
  • Prime-boost vaccination A vaccination method comprising administration of a first immunogenic composition (the primer vaccine) followed by administration of a second immunogenic composition (the booster vaccine) to a subject to elicit an immune response.
  • the primer vaccine and/or the booster vaccine may include a protein antigen, or a vector (such as a viral vector, RNA, or DNA vector) expressing the antigen, and one ore more adjuvants.
  • a suitable time interval between administration of the primer vaccine and the booster vaccine can be determined according to common knowledge in the art.
  • Recombinant Of or resulting from new combinations of genetic material.
  • a recombinant protein is a protein produced by the use of recombinant DNA technology, which involves the combination of genetic material from different sources to create a new (non- naturally occurring) DNA sequence, which is then introduced into a host organism (such as bacteria, yeast, or mammalian cells) to produce the desired protein.
  • Saponin Natural or synthetic glycosides of triterpenes or steroids.
  • Saponins can function as adjuvants.
  • Such saponins include saponins from Quillaja (such as Quil A (a mixture of saponins from Quillaja Saponaria) and QS-21 (a purified saponin from Quillaja Saponaria)), and derivatives and analogs thereof.
  • QS-21 is a water-soluble triterpene glycoside and includes two isomers, QS-21 Apiose (QS- 21 Api) and QS-21 Xylose (QS-21-Xyl), which, in some examples, are present at an about 65:35 ratio.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad.
  • the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at 8123-111083-02 least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984.
  • Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol.
  • the BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.
  • An oligonucleotide is a linear polynucleotide sequence of up to about 100 nucleotide bases in length.
  • reference to “at least 80% identity” refers to “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.
  • T cells A white blood cell critical to the immune response. T cells include, but are not limited to, CD4 + T cells and CD8 + T cells. A CD4 + T lymphocyte is an immune cell that expresses CD4 on its surface.
  • helper T cells help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • Th1 and Th2 cells are functional subsets of helper T cells.
  • Th1 cells secrete a set of cytokines, including interferon-gamma, and whose principal function is to stimulate phagocyte-mediated defense against infections, especially related to intracellular microbes.
  • Th2 cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5, and whose principal functions are to stimulate IgE and eosinophil/mast cell-mediated immune reactions and to downregulate Th1 responses.
  • Toll-like receptor (TLR) agonist A substance that activates a TLR.
  • TLRs belong to a family of innate immune receptors known as pattern recognition receptors. TLRs recognize pathogen-associated molecular patterns (PAMPs) to activate innate immunity and inflammatory cascades, functioning as the first line of defense against pathogens. TLRs also recognize damage-associated molecular patterns (DAMPs) to activate innate immunity to remove damaged or apoptotic cells, and debris.
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damage-associated molecular patterns
  • Transdermal administration refers to delivering one or more substances through an area of skin of a subject, such that the one or more substances pass through the epidermis and reach the microcirculation of the dermis and achieve systemic circulation.
  • the one or more substances may include one or more active ingredients such as an antigen, adjuvant, 8123-111083-02 etc., and may or may not include other non-active ingredients, such as a biocompatible polymer, that assist in formulation of the active ingredients.
  • Microneedle arrays as disclosed herein are for transdermal administration of active ingredients including one or more antigens, a polyphosphazene adjuvant, and optionally a second adjuvant.
  • a microneedle array of the present disclosure will be pressed and held against an area of skin of a subject, such that the microneedles will be inserted into the skin at a depth (e.g., reaching anywhere in the stratum comeum, penetrating the stratum comeum and reaching anywhere in the epidermis, or penetrating the epidermis and reaching anywhere in the dermis before touching nerve endings, dependent on the length of each microneedle) and will break off from the base portion.
  • Tumor An abnormal growth of cells, which can be benign or malignant (a malignancy). Cancer is a malignant tumor (a malignancy), which is characterized by abnormal or uncontrolled cell growth.
  • tumors Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • Metalstatic disease refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor.
  • a tumor that does not metastasize is referred to as “benign.”
  • a tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.”
  • hematological tumors include leukemias, including acute leukemias (such as 11q23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,
  • the lymphoid malignancy can be adult T cell leukemia, cutaneous T cell lymphoma, anaplastic large cell lymphoma, Hodgkin’s lymphoma, or a diffuse large B cell lymphoma.
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, he
  • a tumor is breast, ovarian, gastric or esophageal cancer.
  • Vaccine A preparation of immunogenic material capable of eliciting an immune response, administered for the prevention, inhibition, amelioration, or treatment of an infection or disease caused by a pathogen or parasite.
  • the immunogenic material may include one or more antigens, inactivated pathogens, attenuated pathogens, and/or pathogen- like particles; and one or more adjuvants.
  • a vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen). Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses.
  • Virus-like particle An assembly of molecules that closely resembles a virus, but is non-infectious because it lacks viral genetic material.
  • Viral-like particles can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into virus-like particles.
  • VLPs can be produced using recombinant DNA technology: the genes encoding the viral structural proteins are introduced into a host expression system, such as bacteria, yeast, or mammalian cells; the host cells then produce and assemble the viral structural proteins into VLPs.
  • a host expression system such as bacteria, yeast, or mammalian cells
  • the presently disclosed dissolvable microneedle arrays can be used to induce an immune response to any antigen, including antigens from pathogens and tumor antigens.
  • the antigen can be, for example, a viral antigen, a bacterial antigen, a fungal antigen, or a tumor- associated antigen. 8123-111083-02
  • a DNA encoding the vaccine antigen is utilized.
  • the DNA encodes a viral protein, a bacterial protein, a fungal protein, a parasitic protein, or a tumor associated protein.
  • an mRNA encoding the vaccine antigen is utilized.
  • the mRNA encodes a viral protein, a bacterial protein, a fungal protein, a parasitic protein, or a tumor associated protein.
  • a live attenuated vaccine including the antigen is utilized.
  • the microneedle array can include any one of, but not limited to, peptides, polypeptides, proteins, nuclei acids, polysaccharides, lipids, cells (or components thereof), live-attenuated pathogens (or components thereof), and inactivated pathogens (or components thereof), or pathogen-like particles.
  • An antigen can be derived from a pathogen, e.g., a bacterium, a virus, a parasite, or a fungus, or from a tumor, e.g., a tumor-associated antigen.
  • An antigen from a pathogen can be any molecule or portion of a molecule from a pathogen that can elicit an immune response against the pathogen, which can be produced through recombinant DNA technology or isolated from nature.
  • An antigen from a pathogen can be a subunit antigen, or can be part of a live-attenuated pathogen, inactivated pathogen, or pathogen-like particles.
  • multiple antigens are administered in a single microneedle array to induce protection against multiple diseases, infectious agents, types, serotypes, serovars, and others, and the microneedle arrays of the present disclosure may similarly include multiple antigens.
  • the microneedle array includes an antigen from a bacterium.
  • a vaccine can include a bacterial protein or portion thereof.
  • the protein may be encoded by an mRNA and expressed upon administration to a subject. 8123-111083-02
  • Specific examples of bacterial pathogens include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp.
  • Aeromonas hydrophila Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae
  • Anaplasma phagocytophilum Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans
  • Bacillus sp. such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus
  • Bacteroides sp. such as Bacteroides fragilis
  • Bordetella sp. such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica
  • Borrelia sp. such as Borrelia recurrentis, and Borrelia burgdorferi
  • Brucella sp. such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis
  • Burkholderia sp. such as Burkholderia pseudomallei and Burkholderia cepacia
  • Capnocytophaga sp. Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp.
  • Enterobacter sp such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani
  • Eikenella corrodens Enterobacter sp.
  • Enterobacter aerogenes such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli
  • Enterococcus sp such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani
  • Eikenella corrodens Enterobacter sp.
  • Enterobacter aerogenes such as Enterobacter
  • Ehrlichia sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp.
  • Lactobacillus sp. Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp.
  • Mycobacterium leprae Mycobacterium tuberculosis, Mycobacterium intracellulare, 8123-111083-02 Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum
  • Mycoplasm sp. such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium
  • Nocardia sp. such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis
  • Neisseria sp. such as Neisseria gonorrhoeae and Neisseria meningitidis
  • Pasteurella multocida Plesiomonas shigelloides.
  • Prevotella sp. Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp.
  • Proteus sp. such as Proteus vulgaris and Proteus mirabilis
  • Providencia sp. such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii
  • Pseudomonas aeruginosa Propionibacterium acnes
  • Rhodococcus equi Rickettsia sp.
  • Rhodococcus sp. Rhodococcus sp.
  • Serratia marcescens Stenotrophomonas maltophilia
  • Salmonella sp. such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium
  • Shigella sp. such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei
  • Staphylococcus sp. such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus
  • Streptococcus sp such as Serratia marcesans and Serratia liquifaciens
  • Shigella sp. such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei
  • Staphylococcus sp. such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus
  • Streptococcus pneumoniae for example chloramphenicol- resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, chlor
  • Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia among others.
  • Bacterial antigens suitable for use in a vaccine include proteins, polysaccharides, lipopolysaccharides, and outer membrane vesicles which may be isolated, purified or derived from a bacterium.
  • bacterial antigens include bacterial lysates and inactivated bacteria formulations.
  • Bacterial antigens can be produced by recombinant expression.
  • Bacterial antigens preferably include epitopes which are exposed on the surface of the bacteria during at least one stage of its life cycle.
  • Bacterial antigens include but are not limited to antigens derived from one or more of the bacteria set forth above as well as the specific antigens examples identified below.
  • the vaccine antigen is a gram positive bacterial antigen.
  • the vaccine antigen can be a lipoteichoic acid (LTA).
  • the vaccine antigen is a gram negative bacterial antigen.
  • the vaccine antigen can be a lipopolysaccharide (LPS).
  • Neiserria gonorrhoeae antigens include Por (or porin) protein, such as PorB (see, e.g., Zhu et al. (2004) Vaccine 22:660-669), a transferring binding protein, such as TbpA and TbpB (see, e.g., Price et al. (2004) Infect.
  • Immun.71(l):277-283 an opacity protein (such as Opa), a reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see, e.g., Plante et al. (2000) J. Infect. Dis.182:848-855); WO 99/24578; WO 99/36544; WO 99/57280; and WO 02/079243, all of which are incorporated by reference).
  • Opa opacity protein
  • Rmp reduction-modifiable protein
  • OMV outer membrane vesicle
  • Chlamydia trachomatis antigens include antigens derived from serotypes A, B, Ba and C (agents of trachoma, a cause of blindness), serotypes Li, L3 (associated with Lymphogranuloma venereum), and serotypes, D-K.
  • Chlamydia trachomas antigens also include antigens identified in WO 00/37494; WO 03/049762; WO 03/068811; and WO 05/002619 (all of which are incorporated by reference), including PepA (CT045), LcrE (CT089), Art (CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), MurG (CT761), CT396 and CT761, and specific combinations of these antigens.
  • Treponemapallidum (Syphilis) antigens include TmpA antigen. 8123-111083-02 B.
  • Viral Pathogens the microneedle array includes an antigen from a virus.
  • a vaccine antigen may be a viral protein or portion thereof.
  • the protein may be encoded by an mRNA and expressed upon administration to a subject.
  • viruses include, but are not limited to, a herpesvirus (such as an avian herpesvirus, a bovine herpesvirus, a canine herpesvirus, an equine herpesvirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), Epstein-Barr virus (EBV), an ovine herpesviruses, a porcine herpesvirus, Marek’s disease virus), feline viral rhinotracheitis virus, pseudorabies virus, an avian paramyxovirus, bovine respiratory syncytial virus, human respiratory syncytial virus (RSV), canine distemper virus, canine parainfluenza virus, canine adenovirus, canine parvovirus, monkeypox virus, bovine parainfluenza virus 3, ovine parainfluenza 3, human parainfluenza, rinderpest virus, border disease virus, bovine viral diarrhea virus (BVDV), BVDV Type I, BVDV Type I
  • the virus can be an immunodeficiency virus, such as human immunodeficiency virus (HIV).
  • the virus can be a coronavirus, such as SARS- CoV-1 or SARS-CoV-2.
  • the antigen is a viral surface glycoprotein, or an antigenic (e.g., soluble) fragment thereof.
  • Viral surface glycoproteins are glycoproteins anchoring (e.g., with a transmembrane domain) on the envelope of enveloped viruses.
  • Such proteins include, but are not limited to, glycoprotein (GP) of Filoviridae (e.g., Ebolavirus) and the like; hemagglutinin (HA) and neuraminidase (NA) of Influenza A virus and the like; hemagglutinin (H) and fusion protein (F) of Measles morbillivirus (MeV) and the like; hemagglutinin-neuraminidase (HN) and fusion protein (F) of Mumps orthorubulavirus (MuV) and the like; hemagglutinin-neuraminidase (HN) and fusion protein (F) of human parainfluenza viruses (HPIVs) and the like; spike glycoprotein (S) from coronaviruses and the like; E1 and E2 from Hepacivirus C and the like; G protein and F protein from respiratory syncytial virus (RSV) and the like; G protein and F protein from Henipavirus and the like
  • the antigen is a viral structural protein.
  • these structural proteins include, but are not limited to, capsid proteins, nucleocapsid proteins, matrix proteins, envelope proteins, etc.
  • the viral structural protein is the nucleocapsid protein (N) of SARS-CoV-2.
  • the viral structural proteins are the structural proteins of a Henipavirus (such as a nucleocapsid protein (N), large protein (L), phosphoprotein (P), and matrix protein (M)).
  • the viral structural proteins are the structural proteins of Rotavirus (such as VP1, VP2, VP3, VP4, VP6, and VP7).
  • viral surface glycoprotein antigens can be combined with other viral protein (e.g., viral structural protein) antigens to enhance immune responses (e.g., antibody response and/or cellular response).
  • the virus is a henipavirus.
  • the henipavirus can be a Hendra virus, Nipah virus, or a combination thereof. See, for example, Geisbert et al., npj Vaccines (2021) 6:23 ; doi.org/10.1038/s41541-021-00284-w, incorporated by reference.
  • Hendra virus (HeV) is an enveloped non-segmented negative-sense single-stranded RNA virus of the family Paramyxoviridae.
  • the HeV genome is ⁇ 18,000 nucleotides in length and includes six genes encoding nine proteins, including N (nucleocapsid protein), P (phosphoprotein), M (matrix protein), F (fusion protein), G (attachment protein), and L (RNA polymerase).
  • N nucleocapsid protein
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • G attachment protein
  • L RNA polymerase
  • Exemplary native HeV strain sequences are known, and several models of human HeV infection are available, including model organisms infected with HeV, such as 8123-111083-02 ferrets, mice, golden hamsters, and African Green Monkeys (see, e.g., Rockx, Pathogens and Disease 2014, doi:10.1111/2049-632X.12149, which is incorporated herein by reference in its entirety).
  • Nipah virus is an enveloped non-segmented negative-sense single-stranded RNA virus of the family Paramyxoviridae.
  • NiV genome is ⁇ 18,000 nucleotides in length and includes six genes encoding nine proteins, including N (nucleocapsid protein), P (phosphoprotein), M (matrix protein), F (fusion protein), G (attachment protein), and L (RNA polymerase).
  • Exemplary native NiV strain sequences are known and several models of human NiV infection are available, including model organisms infected with NiV, such as ferrets, mice, golden hamsters, guinea pigs, and African Green Monkeys (see, e.g., Geisbert et al., Curr. Top. Microbiol. Immunol., 359:153-77, 2012, which is incorporated by reference herein in its entirety).
  • the antigenic molecule can be the F (fusion protein), G (attachment protein), or M (matrix protein) of a Hendra virus (such as NiV, HeV, etc), or an antigenic fragment thereof.
  • the antigen can be G (e.g., form NiV or HeV) or an antigenic fragment thereof.
  • the antigen can be a soluble G (e.g., comprising only the ectodomain of G; or lacking the cytoplasmic and transmembrane domains of G).
  • An exemplary G protein from an HeV strain is provided in SEQ ID NO: 1.
  • An exemplary sequence of a soluble G protein (sG) from HeV is provided in SEQ ID NO: 2.
  • an exemplary genome sequence for an HeV strain is provided in GENBANK® Accession No. MN062017, which is incorporated herein by reference.
  • the G proteins of NiV and HeV share about 83% 8123-111083-02 amino acid sequence homology.
  • the antigen can be F (e.g., form NiV or HeV) or an antigenic fragment thereof.
  • F and G are used together in the present microneedle arrays.
  • Soluble forms of Hendra and Nipah Virus G protein that are suitable as antigens for incorporation into the microneedle arrays of the present inventions include, but are not limited to, those described in AU 2005327194 A1; and U.S. Patent Nos.
  • a soluble form of Hendra and Nipah Virus F protein also are suitable as an antigen for incorporation into the microneedle arrays, and include, but are not limited to, those described in AU2013276968A1; and U.S. Patent Nos.10,040,825; and 10,590,172, which are incorporated by references herein in their entireties.
  • a sG includes amino acids 71-604 of a full-length G protein.
  • a sG includes any protein comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 2. 8123-111083-02
  • Epitopes from the HeV sG antigen can be identified using IEDB MHC-II Binding Predictions, MHC-I Binding Predictions, and MHC-I Processing Predictions (www.iedb.org). Identified epitopes can be used to stimulate immune cells in vitro to study their responses. Epitopes can also be used to guide the design of antigenic fragments of an antigen.
  • Exemplary epitopes from HeV sG are provided in Table 2 below.
  • Table 2 Peptide Epitopes from HeV-sG (Uniprot #O89343, HeV-G71-604) Predicted Peptide Sequence Binding 8123-111083-02 442 (SEQ ID NO: 14)
  • SEQ ID NO: 14 Predicted Peptide Sequence Binding 8123-111083-02 442
  • Coronaviruses get their name from the crown-like spikes on their surface.
  • the viral envelope is comprised of a lipid bilayer containing the viral membrane (M), envelope (E) and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illness, such as the common cold.
  • coronavirus is SARS-CoV-2.
  • the antigen is a spike protein, or an antigenic fragment thereof, of the coronavirus, such as SARS-CoV-2.
  • the antigen can be a soluble spike protein (excluding the transmembrane domain and cytoplasmic tail of a spike protein).
  • the antigen can be linked with a bacteriophage T4 fibritin.
  • An exemplary spike protein sequence that can be used as an antigen is provided in SEQ ID NO: 15.
  • a spike protein antigen includes any protein comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 15.
  • An exemplary soluble spike protein sequence that can be used as an antigen is provided in SEQ ID NO: 16.
  • a spike protein antigen includes any protein comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 16.
  • a spike protein antigen includes R683A, R685A, F817P, A892P, A899P, A942P, K986P, and/or V987P mutations with reference to SEQ ID NO: 15 or SESQ ID NO: 16.
  • Ebolavirus is a member of the genus Ebolavirus, which belongs to the family Filoviridae. There are several species of ebolaviruses, including Ebola virus (formerly known as Zaire ebolavirus), Sudan virus (Sudan ebolavirus), Bundibugyo virus (Bundibugyo ebolavirus), Ta ⁇ Forest virus (Ta ⁇ Forest ebolavirus), and Reston virus (Reston ebolavirus). These viruses are known for causing severe and often fatal hemorrhagic fever in humans and other primates. 8123-111083-02 Ebolavirus is a filamentous, enveloped virus within the order Mononegavirales which also contains rabies and measles viruses.
  • -ssRNA single-stranded negative-sense RNA
  • Filoviruses encode seven different proteins that include: NP (nucleoprotein), VP35 (part of the polymerase complex), VP40 (matrix protein), GP (glycoprotein), VP30 (transcription activator), VP42 (second matrix protein), and L (RdRp). Of these proteins, GP and NP proteins are crucial for viral entry and replication.
  • the antigenic molecule can be any of the above protein or an antigenic fragment thereof. In some examples, the antigen is GP or an antigenic fragment thereof.
  • the antigen is a soluble GP (e.g., comprising only the ectodomain of GP).
  • An exemplary GP sequence that can be used as an antigen is provided in SEQ ID NO: 17.
  • a GP antigen includes any protein comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 17.
  • Influenza viruses are a group of RNA viruses belonging to the family Orthomyxoviridae, a family of negative-sense RNA viruses.
  • the family includes seven genera: Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, Deltainfluenzavirus, Isavirus, Thogotovirus, and Quaranjavirus.
  • the first four genera contain viruses that cause influenza in vertebrates (such as birds and mammals, including humans).
  • Species of Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, or Deltainfluenzavirus are called Influenza A viruses, Influenza B viruses, Influenza C viruses, or Influenza D viruses, respectively.
  • the influenzavirus virion is pleomorphic; the viral envelope can occur in spherical and filamentous forms.
  • the virus's morphology is ellipsoidal with particles 100– 120 nm in diameter, or filamentous with particles 80–100 nm in diameter and up to 20 ⁇ m long.
  • the major glycoprotein hemagglutinin (HA) spikes are interposed irregularly by clusters of neuraminidase (NA) spikes, with a ratio of HA to NA of about 10 to 1.
  • the viral envelope composed of a lipid bilayer membrane in which the glycoprotein spikes are anchored encloses the nucleocapsids; nucleoproteins of different size classes with a loop at each end.
  • the ribonuclear proteins are filamentous and fall in the range of 50–130 nm long and 9–15 nm in diameter with helical symmetry.
  • Influenza A viruses are further classified, based on the viral surface proteins hemagglutinin (HA) and neuraminidase (NA).18 HA subtypes (or serotypes) and 11 NA subtypes of influenza A virus have been isolated in nature.
  • the antigenic molecule can be an HA or NA protein, or an antigenic fragment thereof.
  • the antigen is HA or an antigenic fragment thereof.
  • the antigen is a soluble HA (e.g., excluding the transmembrane domain and cytoplasmic tail of an HA).
  • An exemplary HA sequence that can be used as an antigen is provided in SEQ ID NO: 18.
  • an HA antigen includes any protein comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 18.
  • An exemplary soluble HA sequence that can be used as an antigen is provided in SEQ ID NO: 19.
  • an HA antigen includes any protein comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 19.
  • SEQ ID NO: 18 MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLR GVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEI FPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPST SADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPR YAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSI QSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFT AVGKEFNHLEKRIENLNKKV
  • Hepatitis C virus is a small (55–65 nm in size), enveloped, positive-sense single-stranded RNA virus of the genus Hepacivirus, of the family Flaviviridae.
  • the hepatitis C virus is the cause of hepatitis C and some cancers such as liver cancer (hepatocellular carcinoma, abbreviated HCC) and lymphomas in humans.
  • the hepatitis C virus particle includes a lipid membrane envelope that is about 55 to 65 nm in diameter.
  • Two viral envelope glycoproteins, E1 and E2 are embedded in the lipid envelope. They take part in viral attachment and entry into the cell. Within the envelope is an icosahedral core that is about 33 to 40 nm in diameter.
  • HVR1 hypervariable region 1
  • HVR1 hypervariable region 1
  • HVR1 is flexible and quite accessible to surrounding molecules. HVR1 helps E2 shield the virus from the immune system. It prevents CD81 from latching onto its respective receptor on the virus. In addition, E2 can shield E1 from the immune system. Although HVR1 is quite variable in amino acid sequence, this region has similar chemical, physical, and conformational characteristics across many E2.
  • the antigenic molecule can be an E1 and/or E2 protein, or an antigenic fragment thereof.
  • the antigen is a soluble E1 and/or E2 protein (e.g., comprising only the ectodomain of E1 and/or E2), or an antigenic fragment thereof.
  • the antigen is a fusion protein of E1 and E2, or soluble E1 and E2.
  • the antigen is a native-like secreted form of the HCV E1E2 heterodimer, as described in Guest, Johnathan D et al. (“Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer.” Proceedings of the National Academy of Sciences of the United States of America vol.118,3 (2021): e2015149118.
  • the microneedle array includes an antigen from a fungus.
  • a vaccine antigen may be a fungal protein or portion thereof.
  • the protein may be encoded by an mRNA and expressed upon administration to a subject.
  • Exemplary fungal pathogens include one or more of Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such as Candida albicans), Aspergillus sp.
  • the microneedle array includes a vaccine antigen from a parasite.
  • Such a vaccine antigen may be a parasitic protein or portion thereof.
  • the protein may be encoded by an mRNA and expressed upon administration to a subject.
  • Exemplary parasitic organisms include Malaria (Plasmodium falciparum, P. vivax, P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarial nematodes, Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T. solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinella spiralis) or Coccidiosis (Eimeria species).
  • Malaria Plasmodium falciparum, P. vivax, P. malariae
  • Schistosomes Trypanosomes
  • Leishmania Laishmania
  • Filarial nematodes Trichomoniasis
  • Sarcosporidiasis Sarcosporidiasis
  • Taenia T. saginata
  • the microneedle array includes an antigen from a tumor, e.g., a tumor-associated antigen, and a tumor-specific antigen.
  • tumor antigens can be classified into two main categories: tumor-specific antigens, which are endogenously present only on tumor cells, and tumor-associated antigens (TAA), which are present on both tumor cells and some normal cells, and have been effectively targeted in solid and hematopoietic malignancies.
  • a tumor antigen may be a protein or a portion thereof. The protein may be encoded by an mRNA and expressed upon administration to a subject.
  • Exemplary tumor-associated antigens include one or more of the following: Melan-A/MART-1, glycoprotein (gp) 75, gp100, beta-catenin, preferentially expressed antigen of melanoma (PRAME), MUM-1, Wilms tumor (WT)-1, carcinoembryonic antigen (CEA), PR-1, b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, 8123-111083-02 thyroglobulin, RAGE-1, tyrosinase, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, macrophage colony stimulating factor, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin and telomerase, prostate-carcinom
  • Table 1 A list of selected tumors and their associated antigens are shown below in Table 1.
  • Table 1 Exemplary tumors and representative tumor-associated antigens Tumor Tumor-Associated Antigens Acute m elo enous leukemia Wilms tumor 1 (WT1) PRAME PR1 roteinase 3, 8123-111083-02 Additional tumor antigens are known in the art (for example see Novellino et al., Cancer Immunol. Immunother.54(3):187-207, 2005).
  • WT1 Acute m elo enous leukemia Wilms tumor 1
  • PRAME PR1 roteinase 3 8123-111083-02 Additional tumor antigens are known in the art (for example see Novellino et al., Cancer Immunol. Immunother.54(3):187-207, 2005).
  • F. Modified Antigens, Attenuation, and Carrier-formulated Antigens Naturally occurring antigens can be modified to include deletions, additions and/or modifications. These
  • Diphtheria and tetanus toxoids are examples of natural antigens that can be used in a detoxified form, such as produced by chemical (e.g., formaldehyde) treatment.
  • Antigens of use also can be produced using enzymatic digestion/fragmentation of protein antigens, denaturation (commonly through heat or chemical treatment), conjugation, chemical modification, and others.
  • Antigens may also be associated with a carrier protein or particle that mediates the immunogenicity of the antigens.
  • An antigen can be a subunit antigen, or can be part of (or carried by) a live-attenuated pathogen (such as a live attenuated virus or bacterium), inactivated pathogen (such as an inactivated virus or bacterium), or pathogen-like particles.
  • Live-attenuated pathogens are pathogens made less pathogenic through an attenuation process to diminish the pathogen's ability to replicate or cause disease.
  • Attenuation can be achieved through various methods, including serial passaging (repeatedly culturing the pathogen in a way that selects for less virulent strains), irradiation (gamma or ultraviolet rays), genetic manipulation (such as to delete or modify the gene that confers virulence or the ability to replicate in a host), or induced mutation and selection for less virulent strains.
  • Inactivated pathogens are pathogens made nonviable or killed (incapable of replication). Most often, inactivation uses heat, irradiation, or chemical treatment such as formalin or formaldehyde, which disrupts or renders nonfunctional the pathogen’s machinery for replication.
  • inactivated pathogens include increased safety in immunosuppressed hosts and the ability to create vaccines for pathogens that are not readily attenuated. They may also induce fewer undesirable side effects (less reactogenic).
  • Inactivated pathogens are of use in the disclosed microneedle arrays.
  • Pathogen-like particles PLPs are developed to present antigen and adjuvant together in a manner that mimics the pathogen-immune cell interaction. These PLPs are of use in the disclosed microneedle arrays.
  • PLP carriers can be nanoparticles, which can carry an antigen through many ways including chemical bonding (such as covalent conjugation), encapsulation (antigen 8123-111083-02 encapsulated within the nanoparticles, with or without attachment), adsorption (antigen adsorbed on the surface of the nanoparticle), or simple mixing.
  • suitable nanoparticles include polymeric nanoparticles, inorganic nanoparticles, liposomes, immunostimulating complex (ISCOM), self-assembled proteins, and emulsions.
  • Suitable polymeric nanoparticles include poly( D,L -lactide-co-glycolide) (PLG), poly(D,L-lactic-co-glycolic acid)(PLGA), poly(g-glutamic acid) (g-PGA), poly(ethylene glycol) (PEG), polystyrene, or polysaccharide (such as pullulan, alginate, inulin, or chitosan).
  • Suitable inorganic nanoparticles include silica-based nanoparticles (SiNPs) (including porous SiNPs such as mesoporous silica nanoparticles), carbon nanoparticles, gold nanoparticles, or calcium phosphate nanoparticles.
  • Liposomes are formed by biodegradable and nontoxic phospholipids. Liposomes can encapsulate antigen within the core for delivery and incorporate viral envelope glycoproteins to form virosomes.
  • Combination of 1,2-dioleoyl-3- trimethylammonium propane (DOTAP) modified cationic liposome and a cationic polymer (usually protamine) condensed DNA are called liposome-polycation-DNA nanoparticles (LPD), a commonly used adjuvant delivery system in DNA vaccine studies.
  • DOTAP 1,2-dioleoyl-3- trimethylammonium propane
  • LPD liposome-polycation-DNA nanoparticles
  • the components of LPD spontaneously rearrange into a nano-structure around 150 nm in size with condensed DNA located inside the liposome.
  • Liposomes modified with maleimide can be synthesized into interbilayer-crosslinked multilamellar vesicles (ICMVs) by cation driven fusion and crosslinking enabling slowed release of entrapped antigen.
  • ICMVs interbilayer-crosslinked multilamellar vesicles
  • a number of liposome systems have been established and approved for human use, such as Inflexal® V and Epaxal®.
  • ISCOMs are cage like particles about 40 nm large in size, made of the saponin adjuvant, cholesterol, phospholipids, and protein antigen. These spherical particles can trap the antigen by apolar interactions.
  • ISCOMATRIXTM includes ISCOMs without antigen.
  • ISCOMATRIXTM can be mixed with antigen, enabling a more flexible application than is possible for ISCOMs, by removing the limitation of hydrophobic antigens.
  • Ferritin is a protein that can self-assemble into nearly-spherical 10 nm structure. By genetically fusing an antigen (such as influenza virus haemagglutinin) to ferritin, the recombined protein spontaneously assembled into an octahedrally-symmetric particle.
  • the major vault protein (MVP) is another kind of self-assembling protein. Ninety-six units of MVP can self-assemble into a barrel-shaped vault nanoparticle, with a size of approximately 40 nm wide and 70 nm long.
  • Nano-sized emulsions can exist as oil-in-water or water-in-oil forms, where the droplet size can vary from 50 nm to 600 nm. Emulsions can carry antigens inside their core for efficient vaccine 8123-111083-02 delivery or can also be simply mixed with the antigen.
  • MF59TM an oil-in-water emulsion which has been licensed as a safe and potent vaccine adjuvant in over 20 countries.
  • a tailorable nano-sized emulsion (TNE) platform technology has been developed using non-covalent click self-assembly for antigen and drug delivery.
  • An oil-in-water nanoemulsion is formed using designed biosurfactant peptides and proteins.
  • immune-evading PEG and a receptor-specific antibody can be arrayed in a selectively proportioned fashion on the aqueous interface of a nano-sized oil-in-water emulsion.
  • PLPs also include virus-like particles (VLPs).
  • VLPs can be used to elicit an immune response against the virus which the VLP is based on, or can be used as nanocarriers to carry an antigen, to which an immune response is desired.
  • VLPs are formed from the self- assembly of viral capsid proteins into particles that closely mimic the structure of natural virus particles. Depending on the biology of the virus, VLPs can be icosahedral or helical (rod-shaped), and may be enveloped or not. VLPs can occur naturally (e.g., poliovirus empty capsids outside of the cell), and can also be recombinantly produced by expressing the proteins required for VLP production.
  • VLPs self-assemble from a single type of coat protein (e.g., L1 of human papillomavirus)
  • other VLPs require the presence of multiple structural proteins (e.g., bluetongue virus VLPs), or a combination of structural and non- structural proteins (e.g., poliovirus VLPs).
  • Their repetitive surface structure and size of 20– 800 nm make VLPs highly immunogenic, capable of inducing a strong humoral and cellular immune response.
  • VLPs are of use in the disclosed microneedle arrays.
  • VLPs can be engineered to carry antigens (or antigenic epitopes) derived from either the parental virus or foreign sources.
  • the antigens may be presented on the outer surface of VLPs or encapsulated by VLPs
  • the cell membrane coating enveloped VLPs enables the presentation of full-length monomeric or multimeric conformational proteins from related or unrelated viruses anchored on the particle’s surface through transmembrane domains.
  • Examples include HIV VLPs displaying HIV glycoproteins, influenza VLPs displaying three distinct HA subtypes, and Newcastle disease virus (NDV) VLPs carrying respiratory syncytial virus (RSV) F and G proteins.
  • NDV Newcastle disease virus
  • RSV respiratory syncytial virus
  • the possibility of displaying multiple HA subtypes provides the advantage of eliciting an immune response against several influenza virus serotypes, resulting in a broad spectrum of protection.
  • nonenveloped VLPs may be employed to present mainly short peptides.
  • Target epitopes may 8123-111083-02 be either genetically fused to subunit proteins of the VLP to form chimeras or attached to the surface of the VLP by covalent or noncovalent means.
  • VLP chimeras have been extensively explored as vaccine candidates since the mid-1990s.
  • VLPs based on the hepatitis B core antigen (HBcAg) can be used as a chimeric carrier for B cell epitopes.
  • the major immunodominant region (MIR) is the most exposed region of the assembled VLP and the optimal locus for introduction of foreign epitopes from different pathogens.
  • VLPs based on the hepatitis surface antigen can be used to display heterologous antigens from different pathogens.
  • VLPs can also be engineered to present epitopes from tumor-associated antigens (TAAs) (e.g., MAGE- 3, MUC1, PSA, HER2/neu) as therapeutic cancer vaccines.
  • TAAs tumor-associated antigens
  • VLPs can also encapsulate an antigen (such as T cell epitopes), through non-specific encapsulation, electrostatic interaction, protein protein interaction, covalent interaction, etc. See McNeale, Donna et al. “Protein cargo encapsulation by virus-like particles: Strategies and applications.” Wiley interdisciplinary reviews.
  • PLPs also include outer membrane vesicles (OMVs), which are non-living spherical nanostructures that derive from the outer membrane of Gram-negative bacteria.
  • OMVs can be used to elicit an immune response against the bacterium from which the OMV is derived, or can be used as nanocarriers to carry an antigen, to which an immune response is desired.
  • OMVs occur spontaneously in nature in response to stress conditions though at low quantities. The amount of outer membrane or periplasmic proteins packaged into OMVs varies by bacterial species.
  • Escherichia coli packages as little as 0.2% of these proteins whereas Neisseria meningitidis packages as much as 12%.
  • Mechanical disruption includes extraction with a calcium chelator (such as EDTA), sonication, vortexing, etc.
  • a calcium chelator destabilizes the bacterial membrane resulting in OMV release. Sonicating or vortexting bacterial pellets fragments the whole bacterium, and the resulting fragments fuse together to form OMVs.
  • Detergents such as deoxycholate or sodium dodecyl sulfate are also commonly used to obtain OMVs.
  • Heterologous antigens can be expressed in a vesiculating bacterial strain.
  • bacterial species like E. coli, that are more permissive to genetic manipulation and produce OMVs at a higher yield allowing them to be used as a universal delivery system.
  • One commonly used approach to generate OMVs carrying heterologous antigens is by fusing the antigen with a membrane associated protein such as cytolysin A (ClyA, a transmembrane protein enriched on secreted OMVs).
  • a nucleic acid (such as a plasmid) comprising a ClyA gene fused with an antigen-encoding gene can be introduced into the host bacteria which will express the desired antigen on the OMV surface.
  • Another approach is through glycoengineering of the lipopolysaccharide (LPS) O antigen.
  • LPS lipopolysaccharide
  • the genes for the O antigen from a pathogen can be expressed in a non-pathogenic O-antigen mutant bacterial strain, and the heterologous O antigens will be synthesized and attached to the lipid A-core of the host bacteria, and displayed on the outer membrane.
  • the microneedle array includes an mRNA encoding the antigen.
  • a signal peptide is utilized.
  • the endogenous signal peptide of a protein that is an antigen from a pathogen, or a tumor antigen can be replaced with a heterologous signal peptide.
  • the mRNA encodes the native signal peptide and does not encode a heterologous signal peptide.
  • the mRNA encodes a heterologous signal peptide and a protein of interest that is a vaccine antigen.
  • a nucleic acid sequence encoding signal peptide can be 5′ to the nucleic acid sequence encoding the protein of interest, e.g., the antigen from the pathogen or the tumor antigen.
  • a nucleic acid sequence encoding signal peptide can be 3′ to the nucleic acid sequence encoding the protein of interest.
  • a heterologous signal peptide can be 5′ or 3′ to the protein of interest.
  • the mRNA encoding the protein of interest has 5′ and 3′ UTRs.
  • the 5′ UTR is between zero and 3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs.
  • the 5′ and 3′ UTR lengths can be modified as needed to increase translation efficiency following transfection of the transcribed RNA
  • the 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′ UTRs for the gene encoding the protein of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and 8123-111083-02 reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
  • 3′ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5′ UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be designed by adding the 5′ UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but are not required for all RNAs to enable efficient translation.
  • the mRNAs that encode the protein of interest includes a 5′ UTR and/or a 3′ UTR that results in greater mRNA stability and higher expression of the mRNA in the cells.
  • the mRNA includes a Kozak seuqence in the 5’ UTR.
  • the Kozak sequence can be, for example, ACCAUGG. This Kozak sequence can be included in any of the 5’ UTRs listed herein.
  • An exemplary 5’ UTR includes, or consists of: UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAU UCUACUUCUAUUGCAGCAAUUUAAAUcauuucuuuuaaagcaaaagcaauuuucugaaaauuuuca ccauuuacgaacgau (SEQ ID NO: 20)
  • the 5’ UTR includes, or consists of CGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 21)
  • the 5’UTR includes, or consists of: AGGAGGGUUUUUACC (SEQ ID NO: 22)
  • the 3’ UTR includes or consists of: AACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCA ACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCC UAAUAAA
  • the mRNA is polyadenylated.
  • the mRNA includes a poly-A tail (e.g., a poly-A tail having 50-200 nucleotides, such as 100-200, 150-200 8123-111083-02 nucleotides, or greater than 100 nucleotides), although in some aspects, a longer or a shorter poly-A tail is used.
  • the recombinant mRNA encoding the protein of interest can include a 5’ capping structure.
  • 5′-capping of modified RNA can be completed concomitantly during IVT using the following chemical RNA cap analogs to generate the 5′-guanosine cap structure: 3′-O- Me-m7G(5′)ppp(5′)G; G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, Mass.).
  • 5′-capping of modified RNA may be completed post-transcriptionally using a Vaccinia Vims Capping Enzyme to generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, Mass.).
  • Cap 1 structure can be generated using both Vaccinia ViJ.us Capping Enzyme and a 2′-0 methyl- transferase to generate: m7G(5′)ppp(5′)G-2′-0-methyl.
  • Cap 2 structure can be generated from the Cap 1 structure followed by the 2′-0-methylation of the 5′-antepenultimate nucleotide using a 2′-0 methyl-transferase.
  • Cap 3 structure can be generated from the Cap 2 structure followed by the 2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-0 methyl-transferase. See U.S. Patent No.9,701,965, incorporated herein by reference.
  • a promoter of transcription can be attached to the DNA template, upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 RNA polymerase promoter, as described in U.S. Published Patent Application No.2016/0030527A1, incorporated herein by reference.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA can be prepared using in vitro transcription (IVT). The IVT can be performed using any RNA polymerase as long as synthesis of the mRNA from the DNA template that encodes the RNA is specifically and sufficiently initiated from a respective cognate RNA polymerase promoter and full-length mRNA is obtained.
  • the RNA polymerase is T7 RNA polymerase, SP6 RNA polymerase or T3 RNA polymerase.
  • capped RNA is synthesized co-transcriptionally by using a dinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAPTM T7 Kit or a MESSAGEMAXTM T7 ARCA-CAPPED MESSAGE Transcription Kit; EPICENTRE or CellScript, Madison, Wis., USA). If capping is performed co-transcriptionally, the 8123-111083-02 dinucleotide cap analog can be an anti-reverse cap analog (ARCA).
  • ARCA anti-reverse cap analog
  • RNA molecules are capped (e.g., greater than 80%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% of the population of mRNA molecules are capped).
  • the mRNA can be prepared by polyadenylation of an in vitro- transcribed (IVT) RNA using a poly(A) polymerase (e.g., yeast RNA polymerase or E. coli poly(A) polymerase).
  • a poly(A) polymerase e.g., yeast RNA polymerase or E. coli poly(A) polymerase.
  • the mRNA is polyadenylated during in vitro transcription (IVT) by using a DNA template that encodes the poly(A) tail.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase.
  • E-PAP E. coli polyA polymerase
  • RNA sequence of use in the disclosed methods includes, in 5′ to 3′ order, a Cap, a 5’UTR including a Kozak sequence, a codon optimized sequence encoding the protein of interest, such as an antigen from a pathogen and a poly A tail.
  • the RNA encodes the native signal peptide of the protein of interest.
  • the mRNA has both a cap on the 5′ end and a 3′ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatemeric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3′ UTR results in normal sized mRNA which is effective in eukaryotic transfection when it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3′ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • the conventional method of integration of polyA/T stretches into a DNA template is 8123-111083-02 molecular cloning. If a polyA/T sequence integrated into plasmid DNA can cause plasmid instability in some cells, then this instability can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation. H.
  • the disclosed microneedle arrays can include recombinant mRNA encoding the protein of interest, including RNAs that contain one or more modified nucleosides (termed “modified nucleic acids”), which have useful properties including the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced.
  • modified nucleic acids include RNAs that contain one or more modified nucleosides (termed “modified nucleic acids”), which have useful properties including the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced.
  • modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity.
  • Exemplary mRNA vaccinations include, for example and without limitation, the COMIRNATY® and SPIKEVAX® vaccines against SARS-CoV-2. See Table 2.
  • a vaccine includes modified nucleic acids, such as a recombinant mRNA encoding the protein of interest, and including one, two, or more than two different nucleoside modifications.
  • the modified nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid.
  • modified nucleosides include pyridin-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiomidine, 4-thio-pseudomidine, 2-thio-pseudowidine, 5- hydroxyuridine, 3-methylmidine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudoutidine, 5-propynyl-uridine, 1-propynyl-pseudomidine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taw.inomethyl-2-thio-utidine, 1-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1-methyl-pse
  • modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-di
  • a modified nucleoside is 5′-O-(1-Thiophosphate)-Adenosine, 5′-O- (1-Thiophosphate)-Cytidine, 5′-O-(1-thiophosphate)-Guanosine, 5′-O-(1-Thiophophate)- Uridine or 5′-O-(1-Thiophosphate)-Pseudouridine.
  • the ⁇ - thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked nucleic acids are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza- guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, J-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • the disclosed mRNA can include a modified uridine or 1-methylpseudouridine.
  • mRNA that contain either uridine, or 1-methylpseudouridine in place of uridine the 1- methylpseudouridine-containing mRNA was translated at a higher level or for a longer duration than the mRNA that contained uridine. Therefore, in some aspects, one or more or all of the uridines contained in the mRNA(s) used in the methods disclosed herein is/are 8123-111083-02 replaced by 1-methylpseudouridine (such as by substituting 1-methylpseudouridine-5′- triphosphate in an IVT reaction to synthesize the RNA in place of uridine-5′-triphosphate).
  • the mRNA used in the disclosed methods contains uridine and does not contain 1-methylpseudouridine.
  • the mRNA includes at least one modified nucleoside (e.g., 1-methylpseudouridine (m1 ⁇ ), pseudouridine ( ⁇ ), 5- methylcytosine (m 5 C), 5-methyluridine (m 5 U), 2′-O-methyluridine (Um or m 2′-O U), 2- thiouridine (s 2 U), or N 6 -methyladenosine (m 6 A)) in place of at least a portion of the corresponding unmodified canonical nucleoside (e.g., in place of substantially all of the corresponding unmodified A, C, G, or T canonical nucleoside).
  • modified nucleoside e.g., 1-methylpseudouridine (m1 ⁇ ), pseudouridine ( ⁇ ), 5- methylcytosine (m 5 C), 5-methyluridine (m 5 U), 2′-O-methyluridine (U
  • the mRNA includes at least one modified nucleoside wherein the nucleotide is pseudouridine ( ⁇ ) or 5-methylcytosine (m 5 C). In some aspects, the mRNA includes both pseudouridine ( ⁇ ) and 5-methylcytosine (m 5 C). In other aspects, the mRNA includes 1-methylpseudouridine.
  • a nucleic acid base, sugar moiety, or internucleotide linkage in one or more of the nucleotides of the mRNA that is introduced into a eukaryotic cell in any of the methods disclosed herein can comprise a modified nucleic acid base, sugar moiety, or internucleotide linkage.
  • Nucleic acids encoding for use in accordance with the disclosure may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc.
  • Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P.
  • Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid.
  • the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased.
  • a modification may also be a 5′ or 3′ terminal modification.
  • the nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least about 50% modified nucleotides, at least about 80% modified nucleotides, or at least about 90% modified nucleotides.
  • the modified mRNA can have a stability of between 12-18 hours or more than 18 hours, such as about 24, 36, 48, 60, 72 or greater than 8123-111083-02 about 72 hours. In some aspects, the modified mRNA is stable for about 12 to about 72 hours, such as about 12 to about 48 hours, about 12 to about 36 hours, or about 12 to about 24 hours.
  • the mRNA component is a modified mRNA with modified uridine, such as a 1-methylpseudouridine in place of uridine and a 7mG(5’)ppp(5’)N1mpNp cap.
  • modified uridine such as a 1-methylpseudouridine in place of uridine and a 7mG(5’)ppp(5’)N1mpNp cap.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/RNA compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about ⁇ 20° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a 8123-111083-02 phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • an RNA molecule is encapsulated in a nanoparticle. Methods for nanoparticle packaging are well known in the art, and are described, for example, in Bose S, et al (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells. J. Virol.78:8146.2004); Dong Y et al.
  • the mRNA is formulated in a lipid nanoparticle for administration to the subject; for example, comprising a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable lipid, or any combination thereof.
  • the lipid nanoparticle is composed of 50 mol% ionizable lipid ((2 hydroxyethyl)(6 oxo 6-(undecycloxy)hexyl)amino)octanoate, 10 mol% 1,2 distearoyl sn glycerol-3 phosphocholine (DSPC), 38.5 mol% cholesterol, and 1.5 mol% 1- monomethoxypolyethyleneglycol-2,3,dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).
  • the mRNA/lipid nanoparticle composition may be provided in any suitable carrier, such as a sterile liquid for injection at a concentration of 8123-111083-02 0.5 mg/mL in 20 mM trometamol (Tris) buffer containing 87 mg/mL sucrose and 10.7 mM sodium acetate, at pH 7.5 and with appropriate diluent.
  • a suitable carrier such as a sterile liquid for injection at a concentration of 8123-111083-02 0.5 mg/mL in 20 mM trometamol (Tris) buffer containing 87 mg/mL sucrose and 10.7 mM sodium acetate, at pH 7.5 and with appropriate diluent.
  • a nucleic acid molecule encoding an antigen can be included in a viral vector, for example, for expression of the immunogen in a host cell, or for immunization of a subject as disclosed herein.
  • the viral vector can be replication-competent.
  • the viral vector can have a mutation in the viral genome that does not inhibit viral replication in host cells.
  • the viral vector also can be conditionally replication-competent.
  • a number of viral vectors have been constructed, that can be used to express an antigen, including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Hopkins et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J.
  • Baculovirus Autographa californica multinuclear polyhedrosis virus; AcMNPV
  • the viral vector can include an adenoviral vector that expresses an antigen.
  • Adenovirus from various origins, subtypes, or mixture of subtypes can 8123-111083-02 be used as the source of the viral genome for the adenoviral vector.
  • Non-human adenovirus e.g., simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses
  • a simian adenovirus can be used as the source of the viral genome of the adenoviral vector.
  • a simian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype.
  • a simian adenovirus can be referred to by using any suitable abbreviation known in the art, such as, for example, SV, SAdV, SAV or sAV.
  • a simian adenoviral vector is a simian adenoviral vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39.
  • a chimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al., Vaccine, 27:1293-1300, 2009).
  • Human adenovirus can be used as the source of the viral genome for the adenoviral vector.
  • Human adenovirus can be of various subgroups or serotypes.
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype.
  • subgroup A e.g., serotypes 12, 18, and 31
  • subgroup B e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50
  • subgroup C e.g., serotypes 1, 2, 5, and 6
  • subgroup D e.g
  • replication-deficient adenoviral vectors examples include multiply replication-deficient adenoviral vectors, are disclosed in U.S. Patent Nos.5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, and International Patent Application Nos. WO 94/28152, WO 95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO 97/21826, and WO 03/022311.
  • AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome.
  • Both sense and antisense strands of AAV DNA are packaged into AAV capsids with equal frequency.
  • An AAV encoding the antigen is of use.
  • the AAV genome is characterized by two inverted terminal repeats (ITRs) that flank two open reading frames (ORFs).
  • ITRs inverted terminal repeats
  • ORFs open reading frames
  • the first 125 nucleotides of the ITR are a palindrome, which folds upon itself to maximize base pairing and forms a T-shaped hairpin structure.
  • the other 20 bases of the ITR called the D sequence, remain unpaired.
  • the ITRs are cis-acting sequences important for AAV DNA replication; the ITR is the origin of replication and serves as a primer for second-strand synthesis by DNA polymerase.
  • the double-stranded DNA formed during this synthesis which is called replicating-form monomer, is used for a second round of self-priming replication and forms a replicating-form dimer.
  • These double-stranded intermediates are processed via a strand displacement mechanism, resulting in single- 8123-111083-02 stranded DNA used for packaging and double-stranded DNA used for transcription.
  • Located within the ITR are the Rep binding elements and a terminal resolution site (TRS). These features are used by the viral regulatory protein Rep during AAV replication to process the double-stranded intermediates.
  • the ITR is also involved in AAV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008). In some aspects, these elements are included in the AAV vector.
  • the left ORF of AAV contains the Rep gene, which encodes four proteins – Rep78, Rep 68, Rep52 and Rep40.
  • the right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2 and VP3).
  • the right ORF also contains two additional frame- shifted ORFs for the membrane-associated accessory protein (MAAP) and the assembly- activating protein (AAP).
  • MAAP membrane-associated accessory protein
  • AAP assembly- activating protein
  • the AAV capsid contains 60 viral capsid proteins arranged into an icosahedral symmetry. VP1, VP2 and VP3 are present in a 1:1:10 molar ratio (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008). In some aspects, these elements are included in the AAV vector.
  • the AAV vector is packaged in the capsid proteins, which can target specific cell types, such as podocytes, mesangial cells, tubular epithelial cells, collecting duct cells, interstitial cells, and vascular cells.
  • a recombinant adeno-associated virus rAAV is generated having a capsid of interest, and can be used.
  • the capsid includes VP1, VP2, and VP3.
  • a host cell which can be cultured that contains a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein of interest, or fragment thereof; a functional rep gene; a minigene composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene, such as a transgene encoding a therapeutic protein; and sufficient helper functions to permit packaging in the capsid protein.
  • AAV adeno-associated virus
  • ITRs AAV inverted terminal repeats
  • transgene such as a transgene encoding a therapeutic protein
  • the components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans.
  • any one or more of the components may be provided by a stable host cell which has been engineered to contain one or more of the components.
  • a stable host cell will contain the component(s) under the control of an inducible promoter.
  • the component(s) can be under the control of a constitutive promoter.
  • Promoters of use include, but are not limited to, the CMV promoter, the CAG promoter, the CB promoter, the SV40 promoter, the ubiquitin promoter, the EF1 ⁇ 8123-111083-02 promoter, the GAPDH promoter, the PGK promoter, the RSV promoter, and the ⁇ -actin promoter.
  • a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters.
  • the minigene, rep sequences, cap sequences, and helper functions required for producing a rAAV can be delivered to the packaging host cell in the form of any genetic element which transfer the sequences carried thereon.
  • the selected genetic element may be delivered by any suitable method, including those described herein.
  • the methods used to construct vectors include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • rAAV virions methods of generating rAAV virions and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No.5,478,745.
  • the rAAV encoding the antigen is then utilized in the disclosed microneedle arrays.
  • AAV variants have been isolated from adenovirus stocks, or human or nonhuman primate tissues.
  • AAV2, AAV3, AAV5, AAV6 were discovered in human cells, while AAV1, AAV4, AAV7, AAV8, AAV9, AAV10 (AAVrh10), AAV11, AAV12 in nonhuman primate samples.
  • HVRs hypervariable regions
  • tissue tropisms of AAV vectors are also influenced by cell surface receptors, cellular uptake, intracellular processing, nuclear delivery of the vector genome, uncoating, and second-strand DNA conversion.
  • Any type of AAV, encoding the antigen can be used in the disclosed microneedle arrays.
  • Adjuvants The presently disclosed dissolvable microneedle arrays include a polyphosphazene adjuvant, and optionally a second adjuvant.
  • Polyphosphazenes achieve many advantageous effects, compared to other adjuvants, when integrated with the microneedle array delivery system, including significantly enhanced immune responses, and compatibility with a broad range of antigens and antigen-carrying carriers and the base material of the microneedle array.
  • a polyphosphazene is dissolvable in aqueous solutions.
  • a polyphosphazene can be a homopolymer or copolymer.
  • the monomeric units are the same.
  • For a copolymer there can be two, three, four, five, six, etc. types of monomeric units, wherein each type of monomeric units is different with respect to one or both of the side groups.
  • a monomeric unit includes the following structure: , wherein R and R′ can be the same or different.
  • a polyphosphazene includes the following structure: wherein n is any integer within the range of 2 to 500,000, or within any two integers within the range of 2 to 500,000, e.g., from 3 to 500,000, from 4 to 500,000, from 5 to 500,000, from 6 to 500,000, from 7 to 500,000, from 8 to 500,000, from 9 to 500,000, from 10 to 500,000, from 20 to 500,000, from 30 to 500,000, 2 to 400,000, from 3 to 400,000, from 4 to 400,000, from 5 to 400,000, from 6 to 400,000, from 7 to 400,000, from 8 to 400,000, from 9 to 400,000, from 10 to 400,000, from 20 to 400,000, from 30 to 400,000, from 2 to 300,000, from 3 to 300,000, from 4 to 300,000, from 5 to 300,000, from 6 to 300,000, from 7 to 300,000, from 8 to 300, from 2 to 300,
  • R and R′ in each monomeric unit are independently selected from PEG, trifluoroethoxy (-O-CH2-CF3), or an organic group comprising an ionic or ionizable moiety, such as a carboxylic acid group (including any ionic form such as a carboxylate group)(-COOH, -COO ⁇ ).
  • R and R′ are an organic group comprising a carboxylic acid group.
  • R or R′ is a PEG group
  • R′ or R is an organic group comprising a carboxylic acid group.
  • R′ is the same in each monomeric unit, so is R′.
  • R is the same in monomeric units of the same type;
  • R′ is the same in monomeric units of the same type; and
  • R or R′ can thus be categorized into R i and R i ′, R ii and R ii ′, R iii and R iii ′, R iv and R iv ′, Rv and Rv′, etc., wherein the numeric subscript denotes the different types of monomeric units.
  • side groups e.g., R and R′
  • the PEG chains are attached to the phosphorus atoms of the backbone through a - O- or -NH- link.
  • the molecular weight of the PEG chains is from about 500 to about 25,000 g/mol, about 1000 to about 25,000 g/mol, about 3000 to about 25,000 g/mol, about 5000 to about 25,000 g/mol, about 7000 to about 25,000 g/mol, about 9000 to 8123-111083-02 about 25,000 g/mol, about 500 to about 20,000 g/mol, about 1000 to about 20,000 g/mol, about 3000 to about 20,000 g/mol, about 5000 to about 20,000 g/mol, about 7000 to about 20,000 g/mol, about 9000 to about 20,000 g/mol, about 500 to about 15,000 g/mol, about 1000 to about 15,000 g/mol, about 3000 to about 15,000 g/mol, about 5000 to about 15,000 g/mol, about 7000 to about 15,000 g/mol, about 7000 to
  • the molecule is about 500 to about 5,000 g/mol, about 500 to about 6,000 g/mol, about 500 to about 7,000 g/mol, about 500 to about 8,000 g/mol, about 500 to about 9,000 g/mol, or about 500 to about 10,000 g/mol.
  • the molecular weight of the PEG chain is about 10,000 g/mol.
  • the organic carboxylic acid group includes an alkyl (such as a C1 to C20 alkyl), alkenyl (such as a C1 to C20 alkenyl), aryl (such as phenyl), or phenoxy carboxylic acid group.
  • the rest of R 1 -R 5 are independently selected from hydrogen (-H), halo (e.g., fluoro (-F), chloro (-Cl), bromo (-Br), and iodo (-I)), or haloalkyl (e.g., trifluoromethyl (-CF 3 ), trichloromethyl (-CCl 3 ), tribromomethyl (-CBr 3 ), and triiodomethyl (-CI3)).
  • halo e.g., fluoro (-F), chloro (-Cl), bromo (-Br), and iodo (-I)
  • haloalkyl e.g., trifluoromethyl (-CF 3 ), trichloromethyl
  • the phenoxy carboxylic acid group is ortho (o- ), meta (m-), or para (p-).
  • R2 and/or R4 is 8123-111083-02 independently halo (-F, -Cl, -Br, or -I), or haloalkyl (-CF 3 , -CCl 3 , -CBr 3 , or -CI 3 ); and the rest of R1-R5 are hydrogen.
  • the polyphosphazene is ; ); by di(carboxylatophenoxy)phosphazene and di(trifluoroethoxy)phosphazene); fluorinated PCPP (PCPP with the carboxylatophenoxy side groups further substituted with one or more -F and/or -CF 3 , preferably in the ortho-position to the carboxylic acid group); poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene] (a copolymer formed by di(carboxylatoethylphenoxy)phosphazene and di(trifluoroethoxy)phosphazene); fluorinated PCEP (PCEP with the carboxylatoethylphenoxy side groups further substituted with one or more -F and/or -CF 3 , preferably in the ortho-position to the carboxylic acid group); poly[di(carboxylatomethylphenoxy)(trifluoroe
  • polyphosphazene adjuvant is soluble in aqueous solutions.
  • polyphosphazene adjuvant contains cationic counterion selected from the imidazoquinoline family, such as imiquimod, resiquimod, or gardiquimod.
  • water-soluble polyphosphazene adjuvant is ionically 8123-111083-02 crosslinked by multivalent cation, such as calcium or magnesium, to form an ionotropic hydrogel.
  • the ionically cross-linked polyphosphazene adjuvant can be configured in the form of microsphere or nanosphere.
  • the polyphosphazene adjuvant is PCPP, fluorinated PCPP, PEGylated PCPP, poly[di(carboxylatophenoxy)(trifluoroethoxy)phosphazene], or any combination thereof
  • the first pathogen is a bacterium, virus, or fungus, or the first antigen is from a tumor.
  • the polyphosphazene adjuvant is PCEP, fluorinated PCEP, PEGylated PCEP, poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene], or any combination thereof; and the first pathogen is a virus, bacterium, or fungus, or the first antigen is from a tumor.
  • the disclosed microneedle arrays can include a STING Agonist.
  • the cGAS (cyclic GMP-AMP synthase)-STING (STING) pathway is a cytosolic DNA-sensing pathway that drives activation of type I IFN and other inflammatory cytokines in the host immune response, such as against cancers.
  • Stimulator of interferon response cGAMP interactor 1 (STING1, also known as STING), is an endoplasmic reticulum-sessile protein that serves as a signaling hub and adaptor protein in the STING pathway, receiving input from several PRRs, most of which sense ectopic DNA species in the cytosol.
  • STING pathway signaling ensures the production of type I interferon (IFN) in response to invading DNA viruses, bacterial pathogens, as well as DNA leaking from mitochondria or the nucleus (e.g., in cells exposed to chemotherapy or radiotherapy).
  • IFN type I interferon
  • a type I IFN response is involved in the initiation of anticancer immune responses.
  • STING can be activated by several cytoplasmic DNA sensors, including cyclic GMP- AMP synthase (CGAS), Z-DNA binding protein 1 (ZBP1, also known as DAI), DEAD-box helicase 41 (DDX41), interferon-gamma inducible protein 16 (IFI16), LRR binding FLII interacting protein 1 (LRRFIP1), MRE11 homolog, double-strand break repair nuclease (MRE11), and possibly also protein kinase, DNA-activated, catalytic subunit (PRKDC, also known as DNA-PK).
  • CGAS cyclic GMP- AMP synthase
  • ZBP1 Z-DNA binding protein 1
  • DDX41 DEAD-box helicase 41
  • IFI16 interferon-gamma inducible protein 16
  • LRRFIP1 LRR binding FLII interacting protein 1
  • MRE11 homolog MRE11
  • MRE11 double-strand break repair nuclease
  • PRKDC cat
  • cGAMP cyclic GMP-AMP
  • IRF3 interferon regulatory factor 3
  • STING agonists suitable for use in the present disclosure include, without limitation, cyclic dinucleotides (CDNs), such as cyclic AMP-GMP (cGAMP, including 3′,3′-cGAMP, 2′,3′-cGAMP, 3′,5′-cGAMP and 2′,5′-cGAMP), cyclic di-GMP (c-di-GMP), and cyclic di- AMP (c-di-AMP); flavone acetic acid (FAA); 5,6-dimethylxanthenone-4-acetic acid (DMXAA, also known as ASA404 or vadimezan); ADU-S100 (ML RRS2 CDA or MIW815); IACS-8779; IACS-8803; BMS-986301; E7766; GSK3745417; MK-1454; MK- 2118; SB11285; BI-STING (BI 1387446); GSK532; JNJ-4412;
  • CDNs
  • STING agonists include Rp,Rp dithio 2',3' c-di-AMP (e.g., Rp,Rp- dithio c-[A(2',5')pA(3',5')p]), or a cyclic dinucleotide analog thereof; a compound depicted in U.S. Patent Publication No.
  • US2015/0056224 e.g., a compound in FIG.2c, e.g., compound 21 or compound 22
  • c-[G(2',5')pG(3',5')p] a dithio ribose O-substituted derivative thereof, or a compound depicted in FIG.4 of PCT Publication Nos.
  • WO 2014/189805 and WO 2014/189806 c-[A(2',5')pA(3',5')p] or a dithio ribose O-substituted derivative thereof, or is a compound depicted in FIG.5 of PCT Publication Nos.
  • WO 2014/189805 and WO 2014/189806 c-[G(2',5')pA(3',5')p], or a dithio ribose O-substituted derivative thereof, or is a compound depicted in FIG.5 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806; 2'-O-propargyl-cyclic-[A(2',5')pA(3',5')p] (2'-O-propargyl-ML-CDA) or a compound depicted in FIG.7 of PCT Publication No. WO 2014/189806.
  • Other exemplary STING agonists are disclosed, e.g., in PCT Publication Nos.
  • the disclosed microneedle arrays can include a TLR agonist.
  • TLRs are a family of transmembrane receptors expressed by various immune (e.g. macrophages, dendritic cells, lymphocytes) and non-immune (e.g. epithelial cells, fibroblasts) cells. TLRs recognize conserved exogenous and endogenous danger signals known as pathogen- associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).
  • PAMPs pathogen- associated molecular patterns
  • DAMPs damage-associated molecular patterns
  • PAMPs include the bacterial endotoxin lipopolysaccharide (LPS) and viral and bacterial nucleic acids, while DAMPs are released by dead or dying host cells during programmed cell death processes.
  • LPS bacterial endotoxin lipopolysaccharide
  • DAMPs are released by dead or dying host cells during programmed cell death processes.
  • TLRs Of the 10 TLRs expressed in humans, six are found on cell surfaces (TLR1, 2, 4, 5, 6, and 10), and four are localized to endosomes (TLR3, 7, 8, and 9).
  • the former 8123-111083-02 recognizes proteins and lipids, whereas the latter engages nucleic acids. See Rolfo, Christian et al.
  • TLRs Upon binding of the appropriate agonists by the leucine-rich repeat (LRR) domain, TLRs recruit Toll-interleukin receptor (TIR) domain-containing adaptor proteins such as MyD88 and TRIF, which activate NF- ⁇ B signaling, MAP kinases, and interferon regulatory factors (IRFs).
  • LRR leucine-rich repeat
  • TIR Toll-interleukin receptor
  • TLR1 agonists suitable for use in the present disclosure include, without limitation, Pam3CSK4 (Pam3CysSerLys4), which is a synthetic triacylated lipopeptide.
  • TLR2 agonists suitable for use in the present disclosure include, without limitation, Pam3CSK4, zymosan, macrophage-activating lipopeptide 2 KDa (MALP-2), and Bacillus Calmette-Guérin (BCG), which is a live attenuated variant of Mycobacterium bovis.
  • TLR3 agonists suitable for use in the present disclosure include, without limitation, poly(I:C) and derivatives. Acting on both TLR3 and RIG-I-like receptors (RLRs), the synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly(I:C)) not only triggers the innate immune response, but also induces subsequent activation of adaptive immunity.
  • poly(I:C) suitable for the present use can be high molecular weight, comprised of long strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer.
  • the average size of high molecular weight poly(I:C) can be from about 1.5 kb to about 8 kb.
  • poly(I:C) suitable for the present use can be low molecular weight, comprised of short strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer.
  • the average size of low molecular weight poly(I:C) can be from about 0.2 kb to about 1.5 kb.
  • Other variants of poly(I:C) were developed in order to improve safety, interferon induction, and immunogenicity of the parent molecule.
  • Such poly(I:C) derivates include poly(I:C 12U) (e.g., Ampligen ® , Hemispherx) and poly-ICLC (Hiltonol ® , Oncovir).
  • Poly (I:C 12U) represents a modified variant of poly(I:C) containing mismatched uracil and guanosine residues, whereas poly-ICLC contains poly-L-lysine in carboxymethylcellulose, which increases resistance against nucleases and thereby enhances and prolongs stability.
  • poly(I:C 12U) is comprised of a single polypurine (inosine) strand hydrogen- 8123-111083-02 bonded with a single polypyrimidine strand (cytosine) containing an inosine H-bond mismatched pyrimidine (uridine) for every 12 cytosines.
  • TLR4 agonists suitable for use in the present disclosure include, without limitation, monophosphoryl lipid A, which is processed from the LPS of Salmonella Minnesota, glucopyranosyl lipid A in a stable emulsion (GLA-SE), and BCG.
  • TLR5 agonists suitable for use in the present disclosure include, without limitation, flagellin and derivatives, such as entolimod.
  • TLR6 agonists suitable for use in the present disclosure include, without limitation, MALP-2.
  • TLR7 agonists suitable for use in the present disclosure include, without limitation, imidazoquinoline compounds (such as resiquimod (R-848), imiquimod, gardiquimod, etc.). Studies have shown that resiquimod stimulates DC maturation by inducing a Th1 cytokine profile and enhancing costimulatory molecule expression. This leads to a more efficient cross-presentation of exogenous antigens and stronger antigen-specific CD8 + T cell responses.
  • TLR7 The synthetic molecule imiquimod superficially resembles a nucleoside analogue but lacks the fourth nitrogen atom present in purines, and instead contains an imidazoquinoline and isobutyl group. Binding of TLR7 by imiquimod induces secretion of pro-inflammatory cytokines, predominantly IFN- ⁇ , TNF- ⁇ and IL-12. This creates a local cytokine milieu biased towards a Th1-type response, with the generation of cytotoxic effectors.
  • TLR8 agonists suitable for use in the present disclosure include, without limitation, imidazoquinoline compounds (such as resiquimod (R-848), imiquimod, gardiquimod, etc.).
  • TLR9 agonists suitable for use in the present disclosure include, without limitation, CpG oligodeoxynucleotides (ODNs), MGN1703, SD-101, and IC31.
  • ODNs CpG oligodeoxynucleotides
  • MGN1703 MGN1703
  • SD-101 SD-101
  • IC31 IC31
  • synthetic ODNs containing unmethylated CpG dinucleotides have been developed.
  • At least three distinct classes of CpG ODN have been identified based on differences in structure and mechanism of the immune response: (i) K-type ODNs (also known as B-type, such as CpG 7909, CpG 1018), (ii) D-type ODNs (also referred to as A-type), and (iii) C-type ODNs.
  • ODNs characteristically trigger immune responses through the activation of 8123-111083-02 plasmacytoid DCs (pDCs) and induction of cytokines such as TNF- ⁇ and IFN- ⁇ . Furthermore, ODNs can enhance antibody responses. Structural differences in the individual ODN-classes feature different characteristics that may be relevant for vaccine development. For example, K-type ODNs, which carry 1 to 5 CpG motifs on a phosphorothioate backbone display improved resistance to nuclease digestion and an extended in vivo half-life. MGN1703 is a synthetic, covalently closed DNA molecule.
  • SD-101 is a synthetic CpG ODN that stimulates pDCs through TLR9 engagement, which causes them to release IFN- ⁇ and mature into efficient APCs.
  • ODNs based on poly(I:C) were designed, creating a novel single-stranded molecule ODN1a, consisting of dimeric repeats of deoxy-Inosine/deoxy-Cytosine linked by an unmodified phosphodiester backbone.
  • ODN1a was further combined with the antimicrobial peptide KLKL5KLK, creating the promising novel adjuvant IC31, which signals via the TLR9/MyD88-dependent pathway of the cellular and humoral immune response.
  • the disclosed microneedle arrays can include a saponin.
  • Saponins are a large family of amphiphilic glycosides of steroids and triterpenes found in plants. Saponins suitable for use in the present disclosure include, without limitation, quillaja saponins (including Quil A, QS-7, QS-17, QS-18, and QS-21), ginseng saponins, astragalus saponins, achyranthes saponins, soybean saponins, and any synthetic or semi-synthetic analogs thereof. The bark extract of the tree Quillaja saponaria was found to have adjuvant activity.
  • Quil A a fraction derived from the extract, is a heterogeneous mixture of approximately 25 different saponins, and has been used successfully for veterinary applications though generally considered too toxic for human use.
  • a number of individual saponins have been purified from the bark extract and found to exhibit adjuvant activity, including QS-7, QS-17, QS-18, and QS-21.
  • QS-21 stands out owing to its unique profile of immunostimulating activity, inducing a balanced Th1/Th2 immunity, which is valuable to a broad scope of applications in combating various microbial pathogens, cancers, and other diseases.
  • QS-21 has also been extensively studied in the form of a combination adjuvant.
  • GPI-0100 is prepared from Quil A by complete removal of the acyl side chain and subsequent incorporation of a dodecylamine chain via amide formation.
  • the resulting complex mixture, GPI-0100 retains the capacity of stimulating humoral and cellular immunity, and is more potent and less toxic than QS-21 under certain circumstances.
  • Immune-stimulating complexes are spherical open cage-like structures (typically 40 nm in diameter) that are spontaneously formed when mixing together cholesterol, phospholipids and Quillaja saponins (such as Quil A) under a specific stoichiometry.
  • ISCOM-based vaccines have been shown to promote both humoral and cellular immune responses in a variety of experimental animal models.
  • Saponins that have adjuvant activities have also been found in other plants, such as ginseng, astragalus, achyranthes, and soybean. See Rajput, Zahid Iqbal et al. “Adjuvant effects of saponins on animal immune responses.” Journal of Zhejiang University. Science. B vol.8,3 (2007): 153-61. doi:10.1631/jzus.2007.B0153.
  • Sapogenins have been identified from the ginseng plant and extracted from its root.
  • Ginseng extract can enhance human and animal immune responses, including antibody production, proliferative response of blood lymphocytes, and blood polymorphonuclear leukocytes phagocytosis.
  • Astragalus contains numerous triterpene saponins (astragalosides I ⁇ X, isoastragalosides I ⁇ IV and soyasaponin I).
  • Astragalus saponins can increase macrophage activities, T- cell transformation, NK cell activity, interferon production, and phagocytosis.
  • Achyranthes root is a crude drug used as diuretic, tonic and remedy for blood stasis.
  • a dissolvable microneedle array includes a base portion, and a plurality of microneedles extending from the base portion.
  • microneedles can have different 8123-111083-02 portions made from different base materials. Individual microneedles can also have different portions including or not including active components, or including active components at different concentrations.
  • the base portion can be made from the same base material as for the microneedles, or can be made from a different material. The base portion can also include or not include active components.
  • A. Microneedle Structures and Base Materials The microneedle arrays of the present disclosure can have the shape and structures in accordance with the disclosure in, for example, U.S. Published Patent Application Nos. US- 2016-0271381-A1, US-2019-0000966-A1, and US-2022-0241570-A1, which are incorporated herein by reference.
  • Microneedles can take on conical, pyramidal, obelisk, or multifaceted shapes that facilitate insertion into the skin.
  • Microneedles may or may not comprise an undercut feature, which refers to a feature (e.g., an indentation, protrusion, or other geometric shape) that restricts or prevents the withdraw of a molded part having this feature from a conventional one-piece mold in injection molding methods.
  • Systems and methods provided herein can be used to fabricate microneedles with or without undercut features.
  • FIG.1 illustrates schematic representation of exemplary microneedle shapes and structures that are generally suitable for fabrication by spin-casting material into a production mold.
  • FIG.1 The structures in FIG.1 include (a) a generally pyramidal microneedle, (b) a “sharp” pillar type microneedle, (c) a “wide” pillar type microneedle, (d) a “short” pillar type microneedle (having a short pillar section and a longer pointed section), and (e) a “filleted” pillar type microneedle.
  • FIG.2 illustrates schematic representation of exemplary microneedle shapes and structures that have an undercut feature. Four different microneedle structures 10, 12, 14, 16 are shown in a production mold 18 that is formed from a flexible material, such as a flexible elastomer.
  • Microneedle 10 is formed with a dissolvable base portion 20, a dissolvable stem 22, and a dissolvable microneedle tip 24 that is loaded with active components.
  • microneedle 10 is fabricated from a dissolvable base material throughout.
  • the active components such as adjuvant and antigen, can be mixed into the dissolvable base material, but is preferably located at the tip of the needles as shown in FIG.2 to improve delivery efficiency.
  • microneedles 10, 12, 14, 16 can have a pyramid head-shape with a sharp tip and an undercut stem that connects to the base portion through filleted bases. Fillets 32 can provide improved mechanical performance during tissue insertion.
  • Microneedle 12 is formed with a non-dissolvable base portion 26, a non-dissolvable stem 28, and a dissolvable microneedle tip 24 that is loaded with a bioactive material.
  • This functionally-graded, undercut microneedle array design can be fabricated with more than one base material.
  • the pyramid portion can be created using a dissolvable base material, while the stem portion and the base portion are manufactured from a non- dissolvable base material, such as a non-dissolvable biocompatible rigid polymer (e.g., poly(methyl methacrylate), polycarbonate, VeroWhite, light (e.g., UV) curable resins, and heat curable resins).
  • a non-dissolvable biocompatible rigid polymer e.g., poly(methyl methacrylate), polycarbonate, VeroWhite, light (e.g., UV) curable resins, and heat curable resins.
  • Microneedle 12 can, therefore, provide a sharp needle tip along with enhanced mechanical performance through a filleted base that enables successful tissue penetration, and a pyramid head that serves as the active component dosage form where the active component(s) are incorporated into a dissolvable matrix.
  • the undercut stem portion improves the mechanical performance during penetration while ensuring tissue retention during implantation and the non-dissolvable nature prevents back diffusion of the embedded active component(s) during both fabrication and implantation processes.
  • the non-dissolvable base portion can also help prevent absorption of humidity during storage, which may result in excessive curvature of the base portion and render applications suboptimal.
  • Microneedle 14 is similar in shape to microneedle 12, but further includes another dissolvable layer 30.
  • the pyramid portion is created using a dissolvable material and a more quickly dissolvable layer is provided adjacent to the stem/needle tip connection.
  • the dissolvable layer can be formed for example, from a small molecular weight quickly dissolvable polymer such as glucose, sucrose, trehalose, maltodextrin, or polyvinylpyrrolidone.
  • the rest of the stem portion and the base portion can be formed from a non-dissolvable base material, such as a non-dissolvable biocompatible rigid polymer such as acrylated polyesters, epoxies, UV-curable monomers, resins, silicones.
  • Microneedle 14 therefore provides all the benefits and features of microneedle 12, and additionally a quickly dissolving layer along with the mechanical mismatch between dissolving and non-dissolving layers that facilitates quick separation of pyramid tips.
  • Microneedle 16 is similar to microneedle 14 but further includes a conforming base portion 34, which may be non-dissolvable.
  • microneedle 16 can be formed with a pyramid portion that is created using a dissolvable material, the quickly dissolving layer can 8123-111083-02 be formed as described above, the rest of the stem portion can be manufactured from a non- dissolvable material, and the base portion can be manufactured from a conformable material, such as a non-dissolvable conformable polymer (silicones, UV-curable polymers, elastomers, etc.).
  • a conformable material such as a non-dissolvable conformable polymer (silicones, UV-curable polymers, elastomers, etc.).
  • Microneedle 16 can therefore provide all the benefits and features of microneedle 14, and additionally a base portion that can conform to non-uniform skin topography better. It should be understood that any combination of the features disclosed in FIG.2—and throughout this application—is contemplated.
  • the conformable base portion of microneedle 16 and the quickly dissolving layer 30 of microneedles 14, 16 can be used in combination with any of the other structures disclosed herein.
  • Dissolvable base materials that can be used to fabricate the microneedle arrays of the present disclosure include, without limitation, carboxymethylcellulose (CMC), trehalose, polyvinylpyrrolidone, poly(vinyl alcohol), maltodextrin, silk, glucose, hyaluronic acid, poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid), sucrose, gelatin, glucose, lactose, collagen, chitosan, poly- ⁇ -glutamate, polyethylene glycol (PEG), or any combination thereof.
  • CMC carboxymethylcellulose
  • trehalose polyvinylpyrrolidone
  • poly(vinyl alcohol) poly(vinyl alcohol)
  • maltodextrin silk
  • glucose hyaluronic acid
  • PLGA poly(lactic-co-gly
  • the base portion of a microneedle array is preferably flat. In some aspects, the base portion is rigid, while in some other aspects, the base portion is conformable.
  • Microneedle arrays incorporating active components, such as adjuvant an antigen, in the entire array can have less delivery efficiency compared to microneedle arrays incorporating active components only in the portions that enter the skin.
  • Systems and methods provided herein enable various active components to be incorporated into the microneedles (or portions of the microneedles, such as the upper portions, tips, etc.), but not the base portion, of the microneedle array. By localizing the active components in this manner, the remainder of the microneedle array can be prepared using less expensive base material.
  • Different portions of individual microneedles can be made to comprise or not comprise an active component, or comprise an active component at different concentrations.
  • an active component is only present in the microneedles but not in the base portion.
  • an active component is concentrated in the upper halves, tips, or portions that taper near the tips of the microneedles, and is not present or present at a trivial amount in the reset of the array.
  • an active component is distributed 8123-111083-02 evenly throughout individual microneedles.
  • the entire array including the base portion includes an active component.
  • a dissolvable microneedle array comprising a base portion, and a plurality of microneedles extending from the base portion.
  • the plurality of microneedles can include different or the same components, and dependent on their compositions, can be divided or categorized into a first, second, third, etc. plurality of microneedles.
  • Each plurality of microneedles may include an effective amount of active components, in order to elicit an immune response.
  • the dissolvable microneedle array is used to prevent (or treat) an infection or disease caused by a pathogen.
  • the dissolvable microneedle array is used to prevent (or treat) a tumor.
  • the dissolvable microneedle array includes only a first plurality of microneedles.
  • an effective amount is the amount in the first plurality to achieve a desired result (e.g., elicit an immune response, and/or prevent an infection or disease).
  • the first plurality of microneedles includes an amount of a first antigen from a first pathogen or tumor (or a nucleic acid molecule encoding the first antigen), and a polyphosphazene adjuvant, effective to elicit an immune response against the first antigen (or first pathogen or tumor).
  • the first plurality of microneedles includes a first antigen from a first pathogen or tumor (or a nucleic acid molecule encoding the first antigen), a second antigen (from the first pathogen or tumor, or from a second pathogen or tumor) (or a nucleic acid molecule encoding the second antigen), and a polyphosphazene adjuvant, effective to elicit an immune response against the first antigen and second antigen (or first pathogen and second pathogen, or first tumor or second tumor).
  • the array has more than one plurality of microneedles (such as a second, third, etc. plurality of microneedles).
  • the dissolvable microneedle array includes a second plurality of microneedles, wherein the second plurality of microneedles includes the same antigen as in the first plurality of microneedles.
  • the second plurality of microneedles includes a different adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin).
  • an array may include a first plurality including a first antigen and a polyphosphazene adjuvant, and a second plurality including the first antigen and a TLR agonist (e.g., poly(I:C)).
  • the active components in the first plurality, 8123-111083-02 combined with the active components in the second plurality are effective to achieve the desired result, e.g., eliciting an immune response to the antigen (or to the first pathogen or tumor).
  • the dissolvable microneedle array includes a second plurality of microneedles, wherein the second plurality of microneedles includes a second antigen different from the first antigen.
  • an array may include a first and a second pluralities of microneedles, wherein the amount of the first antigen and an adjuvant in the first plurality of microneedles is effective to elicit an immune response to the first antigen, and the amount of the second antigen and an adjuvant in the second plurality is effective to elicit an immune response to the second antigen, when the dissolvable microneedle array is administered to a subject.
  • the first and second antigens can be from the same pathogen or tumor.
  • the first and second antigens can be from a different pathogen or tumor.
  • the dissolvable microneedle array can be used to elicit an immune response to a first pathogen and second pathogen, respectively.
  • the dissolvable microneedle array can be used to elicit an immune response to this pathogen.
  • the dissolvable microneedle array can be used to elicit an immune response to these two tumors.
  • both the first plurality of microneedles and the second plurality of microneedles include a polyphosphazene adjuvant.
  • each microneedle of the first plurality includes a polyphosphazene adjuvant, and a first antigen from a first pathogen (or tumor) or a nucleic acid molecule encoding the first antigen.
  • the first plurality of microneedles includes an amount of the polyphosphazene adjuvant and the first antigen effective to elicit an immune response to the first antigen or pathogen in a subject.
  • each microneedle of the second plurality includes a polyphosphazene adjuvant, and a second antigen from the first pathogen (or tumor) or second pathogen (or tumor) or a nucleic acid molecule encoding the second antigen.
  • the second plurality of microneedles includes an amount of the polyphosphazene adjuvant and the second antigen effective to elicit an immune response to the second antigen or pathogen in a subject.
  • only the first, or only the second, plurality of microneedles includes a polyphosphazene adjuvant
  • the second, or the first, plurality of microneedle, respectively includes a different adjuvant
  • the dissolvable microneedle array includes a first plurality of microneedles including an amount of a polyphosphazene adjuvant and a first antigen from a first pathogen (or a tumor), and a second plurality of microneedles including an amount of a second antigen from the first pathogen (or tumor), and a second adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin) effective to elicit an immune response to the first pathogen (or tumor) in a subject.
  • a second adjuvant a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin
  • the dissolvable microneedle array includes a first plurality of microneedles including an amount of a polyphosphazene and an antigen from a first pathogen (or a tumor), and a second plurality of microneedles including an amount of an antigen from a second pathogen (or tumor), and a second adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin) effective to elicit an immune response to the first pathogen (or tumor) and the second pathogen (or tumor) in a subject.
  • a second adjuvant a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin
  • the polyphosphazene adjuvant and the antigen are present at a ratio (weight/weight (w/w)) of about 1:1 to about 100:1, about 1:1 to about 90:1, about 1:1 to about 80:1, about 1:1 to about 70:1, about 1:1 to about 60:1, about 1:1 to about 50:1, about 1:1 to about 40:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 1:1 to about 20:1, about 1:1 to about 15:1, about 2:1 to about 100:1, about 2:1 to about 90:1, about 2:1 to about 80:1, about 2:1 to about 70:1, about 2:1 to about 60:1, about 2:1 to about 50:1, about 2:1 to about 40:1, about 2:1 to about 30:1, about 2:1 to about 25:1, about 2:1 to about 20:1, about 2:1 to about 15:1, about 3:1 to about 100:1, about 3:1 to about 90:1, about 3:1 to about 80:1, about 3:1 to about 70:1, about 3:1 to about 60:1, about 3:1 to about 50:1, about 3:1 to about
  • each microneedle including polyphosphazene adjuvant(s) and antigen(s) includes the polyphosphazene adjuvant(s) and the antigen(s) at the aforementioned ratios.
  • the polyphosphazene adjuvant can be any of those disclosed above.
  • the polyphosphazene adjuvant includes at least one side group including a carboxylic acid.
  • the side group is a phenoxy group substituted with a carboxylic acid.
  • the phenoxy group is an ortho (o-), meta (m-), or para (p- ) phenoxy group.
  • the carboxylic acid is–(CH 2 ) m –COO ⁇ , wherein m is 0 or any integer from 1-10.
  • the phenoxy group is further substituted with a fluorine or a trifluoromethyl group.
  • the polyphosphazene adjuvant including at least one side group which is a phenoxy group with a –(CH2)m–COO ⁇ substituent, wherein m is 0 or any integer from 1-10.
  • the polyphosphazene adjuvant is PCPP, fluorinated PCPP, PEGylated PCPP, PCEP, fluorinated PCEP, PEGylated PCEP, PCMP, fluorinated PCMP, PEGylated PCMP, poly[di(carboxylatophenoxy)(trifluoroethoxy)phosphazene], poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene], poly[di(carboxylatomethylphenoxy)(trifluoroethoxy)phosphazene], or any combination thereof.
  • the antigen can be from any pathogen, including bacteria and viruses, or can be a tumor antigen.
  • the virus is a henipavirus (such as Hendra virus 8123-111083-02 and Nipah virus), influenza virus, coronavirus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), ebolavirus, or filovirus.
  • the bacterium is Neisseria meningitidis, Neisseria gonorrhoeae, or Chlamydia. It is disclosed herein that certain polyphosphazene adjuvants can result in skewed immune responses, such as Th1-skewed or Th2-skewed immune responses.
  • polyphosphazene with carboxylatoethylphenoxy side groups such as PCEP, fluorinated PCEP, PEGylated PCEP, poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene], etc.
  • polyphosphazene with carboxylatophenoxy side groups such as PCPP, fluorinated PCPP, PEGylated PCPP, poly[di(carboxylatophenoxy)phosphazene], etc.
  • Th1- skewed immune responses in general are more efficient in eliminating viruses, and Th2- skweded immune response in general are more efficient in eliminating bacteria. Therefore, in some examples, polyphosphazene stimulating a Th1-skewed response (e.g., PCEP, fluorinated PCEP, PEGylated PCEP, or poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene]) is formulated with a virus antigen.
  • PCEP polyphosphazene stimulating a Th1-skewed response
  • fluorinated PCEP e.g., fluorinated PCEP, PEGylated PCEP, or poly[di(carboxylatoethylphenoxy)(trifluoroethoxy)phosphazene]
  • polyphosphazene stimulating a Th2-skewed response e.g., PCPP, fluorinated PCPP, PEGylated PCPP, poly[di(carboxylatophenoxy)phosphazene-co- di(trifluoroethoxy)phosphazene]
  • PCPP polyphosphazene stimulating a Th1-skewed response
  • polyphosphazene stimulating a Th2-skewed response can be combined with polyphosphazene stimulating a Th2-skewed response, to achieve a balanced Th1 and Th2 response.
  • the dissolvable microneedle array includes a base portion, and a first plurality of microneedles extending from the base portion.
  • the microneedle array may include only the first plurality of microneedles or include one or more additional pluralities of microneedles.
  • the first plurality includes an amount of a polyphosphazene adjuvant, and a first antigen from a first pathogen or tumor (or a nucleic acid encoding the first antigen) effective to elicit an immune response to the first antigen or pathogen (or tumor) in a subject.
  • the polyphosphazene adjuvant and the first antigen is present at a ratio (weight/weight (w/w)) described herein.
  • each microneedle in the first plurality includes the polyphosphazene adjuvant and the first antigen at the ratio.
  • the first antigen from the first pathogen is a G glycoprotein (such as a recombinant soluble G glycoprotein) or an antigenic fragment thereof from a henipavirus, such as a Hendra virus or a Nipah virus.
  • the G glycoprotein antigen formulated with polyphosphazene and delivered through a microneedle array elicits potent immune responses that not only protect against infection by the virus which the antigen is from, but also protect 8123-111083-02 against infection by other virus species within the same genus.
  • the use of the disclosed microneedle array including a polyphosphazene adjuvant and an antigen produces a cross-reactive immune response.
  • the first plurality of microneedles further includes an amount of a second adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin) effective to enhance the immune response to the first pathogen in the subject.
  • a second adjuvant a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin
  • the second adjuvant associates with the polyphosphazene adjuvant (through ionic or electrostatic interaction) as a counterion.
  • the polyphosphazene adjuvant and the second adjuvant are present at a ratio (w/w) of about 1:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 2:1 to about 10:1, about 2:1 to about 9:1, about 2:1 to about 8:1, about 2:1 to about 7:1, about 2:1 to about 6:1, about 2:1 to about 5:1, about 2:1 to about 4:1, about 3:1 to about 10:1, about 3:1 to about 9:1, about 3:1 to about 8:1, about 3:1 to about 7:1, about 3:1 to about 6:1, about 3:1 to about 5:1, about 3:1 to about 4:1, about 4:1 to about 10:1, about 4:1 to about 9:1, about 4:1 to about 8:1, about 4:1 to about 7:1, about 4:1 to about 6:1, about 4:1 to about 5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about or 10:1
  • the first plurality of microneedles further includes an amount of a second antigen (from the first pathogen or a second pathogen) effective to elicit an immune response to the second antigen (or second pathogen) when delivered together with the amount of adjuvants in the first plurality of microneedles.
  • the second antigen is a henipavirus F protein.
  • the second antigen is a soluble henipavirus F protein.
  • the dissolvable microneedle array further includes a second plurality of microneedles extending from the base portion, including an amount of the first antigen from the first pathogen (or tumor), and a second adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin), which when delivered together with the amount of active components present in the first plurality, effective to elicit an immune response to the first antigen or pathogen (or tumor) in a subject.
  • each microneedle in the second plurality comprises the first antigen and the second adjuvant.
  • the dissolvable microneedle array further includes a second plurality of microneedles extending from the base portion, including an amount of a second antigen (different from the first antigen) from the first pathogen (or tumor), and a polyphosphazene adjuvant effective to elicit an immune response to the second antigen in a subject.
  • a second antigen different from the first antigen
  • the polyphosphazene adjuvant and the second antigen are present at a ratio (weight/weight (w/w)) described herein.
  • each microneedle in the second plurality includes the polyphosphazene adjuvant and the second antigen at the ratio.
  • the dissolvable microneedle array further includes a second plurality of microneedles extending from the base portion, including an amount of a second antigen (different from the first antigen) from the first pathogen (or tumor), and a second adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin) effective to elicit an immune response to the second antigen in a subject.
  • a second antigen different from the first antigen
  • a second adjuvant a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin
  • the dissolvable microneedle array further includes a second plurality of microneedles extending from the base portion, including an amount of a second antigen from a second pathogen (or tumor, different from the first pathogen, or tumor), and a polyphosphazene adjuvant, effective to elicit an immune response to the second antigen, pathogen, or tumor in a subject.
  • the polyphosphazene adjuvant and the second antigen are present at a ratio (weight/weight (w/w)) described herein.
  • each microneedle in the second plurality includes the polyphosphazene adjuvant and the second antigen at the ratio.
  • the dissolvable microneedle array further includes a second plurality of microneedles extending from the base portion, including an amount of a second antigen from a second pathogen (or tumor, different from the first pathogen, or tumor), and a second adjuvant (a TLR agonist, a cGAS/STING pathway agonist, and/or a saponin) effective to elicit an immune response to the second antigen, pathogen, or tumor in a subject.
  • each microneedle in the second plurality includes the second antigen and the second adjuvant.
  • the above described second pluralities can be included, in any combination, in one microneedle array to form a third, fourth, fifth, etc. pluralities of microneedles.
  • the microneedles of the first, second, and/or any additional pluralities of microneedles comprise a dissolvable biocompatible material.
  • the polyphosphazene adjuvant and the dissolvable biocompatible material in the first, second, and/or any additional pluralities of microneedles are present at a ratio (weight/weight (w/w)) ranging from about 1:1 to about 1:150, about 1:1 to about 1:125, about 1:1 to about 1:100, about 1:10 to about 1:150, about 1:10 to about 1:125, about 1:10 to about 1:100, about 1:20 to about 1:150, about 1:20 to about 1:125, about 1:20 to about 1: 100, about 1:30 to about 1:150, about 1:30 to about 1:125, about 1:30 to about 1:100, about 1:40 to about 8123-111083-02 1:150, about 1:40 to about 1:125, about 1:40 to about 1:100, about 1:50 to about 1:150, about 1:50 to about 1:125, about 1:50 to about 1:100.
  • the dissolvable biocompatible material is used as a base material in providing structure to the microneedles.
  • Such structural substrates include, without limitation, carboxymethylcellulose (CMC), trehalose, polyvinylpyrrolidone, poly(vinyl alcohol), maltodextrin, silk, glucose, hyaluronic acid, poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid), sucrose, gelatin, glucose, lactose, collagen, chitosan, poly- ⁇ -glutamate, polyethylene glycol (PEG), or any combination thereof.
  • CMC carboxymethylcellulose
  • trehalose polyvinylpyrrolidone
  • poly(vinyl alcohol) poly(vinyl alcohol)
  • maltodextrin silk
  • glucose hyaluronic acid
  • PLGA poly(lactic-co-glycolic acid)
  • PEG polyethylene glycol
  • microneedle arrays With various microneedle shapes and structures described above, and capable of simultaneous delivery of multiple chemically distinct active components, such as multiple antigens (or nucleic acids encoding the antigens) and adjuvants, to generate potent immune responses.
  • the microneedle arrays of the present disclosure can be fabricated in accordance with the methods disclosed in, for example, U.S. Published Patent Application Nos. US-2016- 0271381-A1, US-2019-0000966-A1, and US-2022-0241570-A1, which are incorporated herein by reference.
  • Micromachining of Master Molds and Creation of Production Molds can be performed to create microneedle arrays of various specifications.
  • apparatuses and methods are described for fabricating dissolvable microneedle arrays using master molds formed by micromilling techniques.
  • Micromilling technology can be used to generate various micro-scale geometries on virtually any type of material, including metal, polymer, and ceramic parts.
  • Micromilled mastermolds of various shapes and configurations can be effectively used to generate multiple identical female production molds.
  • the female production molds can then be used to micro-cast various microneedle arrays.
  • Mechanical micromilling uses micro-scale (for example, as small as 10 ⁇ m) milling tools within precision computer controlled miniature machine-tool platforms.
  • the system can include a microscope to view the surface of the workpiece that is being cut by the micro- tool.
  • the micro-tool can be rotated at ultra-high speeds (200,000 rpm) to cut the workpiece to create the desired shapes.
  • the micromilling process can be used to create 8123-111083-02 complex geometric features with many kinds of material.
  • Various types of tooling can be used in the micromilling process, including, for example, carbide micro-tools.
  • diamond tools can be used to fabricate the microneedle arrays on the master mold. Diamond tooling can be preferable over other types of tooling because it is harder than conventional materials, such as carbide, and can provide cleaner cuts on the surface of the workpiece.
  • Mastermolds can be micromilled from various materials, including, for example, CIRLEX® (DuPont, Kapton® polyimide). Mastermolds can be used to fabricate flexible production molds from a suitable material, such as SYLGARD® 184 (Dow Corning). The mastermold is desirably formed of a material that is capable of being reused so that a single mastermold can be repeatedly used to fabricate a large number of production molds. Similarly, each production mold is desirably able to fabricate multiple microneedle arrays. Mastermolds can be created relatively quickly using micromilling technology.
  • a mastermold that includes a 10 mm x 10 mm array with 100 microneedles can take less than a couple of hours and, in some aspects, less than about 30 minutes to micromill.
  • a short ramp-up time enables rapid fabrication of different geometries, which permits the rapid development of microneedle arrays and also facilitates the experimentation and study of various microneedle parameters.
  • the mastermold material preferably is able to be cleanly separated from the production mold material and preferably is able to withstand any heighted curing temperatures that may be necessary to cure the production mold material.
  • the silicone-based compound SYLGARD® 184 (Dow Corning) is the production mold material and that material generally requires a curing temperature of about 80-90 degrees Celsius.
  • Mastermolds can be created in various sizes. For example, a mastermold was created on 1.8 mm thick CIRLEX® (DuPont, Kapton® polyimide) and 5.0 mm thick acrylic sheets. Each sheet can be flattened first by micromilling tools, and the location where the microneedles are to be created can be raised from the rest of the surface. Micro-tools can be used in conjunction with a numerically controlled micromilling machine to create the microneedle features (e.g., as defined by the mastermold). In that manner, the micromilling process can provide full control of the dimensions, sharpness, and spatial distribution of the microneedles.
  • CIRLEX® DuPont, Kapton® polyimide
  • Micro-tools can be used in conjunction with a numerically controlled micromilling machine to create the microneedle features (e.g., as defined by the mastermold). In that manner, the micromilling process can provide full control of the dimensions, sharpness, and spatial distribution of the microneedles.
  • FIG.3 is an image from a scanning electron microscope (SEM) showing the structure of a micromilled mastermold with a plurality of pyramidal needles.
  • SEM scanning electron microscope
  • a circular groove can be formed around the microneedle array of the mastermold to produce an 8123-111083-02 annular (for example, circular) wall section in the production mold.
  • the circular wall section of the production mold can facilitate the spin-casting processes discussed below.
  • wall sections or containment means of other geometries can be provided.
  • the containment means can be formed in a variety of shapes including, for example, square, rectangular, trapezoidal, polygonal, or various irregular shapes.
  • the production molds can be made from SYLGARD® 184 (Dow Corning), which is a two component clear curable silicone elastomer that can be mixed at a 10:1 SYLGARD® to curing agent ratio.
  • the mixture can be degassed for about 10 minutes and poured over the mastermold to form an approximately 8 mm layer, subsequently degassed again for about 30 minutes and cured at 85 °C for 45 minutes.
  • the mastermold can be separated from the cured silicone, and the silicone production mold trimmed to the edge of the circular wall section that surrounds the array (FIG.4A).
  • FIG.4A is an SEM image of a pyramidal production mold created as described above.
  • FIG. 4B illustrates an enlarged segment of the production mold with a pyramidal needle molding well in the center of the image.
  • the molding well is configured to receive a base material (and any components added to the base material) to form microneedles with an external shape defined by the molding well.
  • Additive Manufacturing of Master Molds and Creation of Production Molds Microneedle arrays can also be formed using additive manufacturing (AM), including micro-additive manufacturing ( ⁇ AM) techniques.
  • AM additive manufacturing
  • ⁇ AM micro-additive manufacturing
  • CAD computer-aided design
  • Exemplary manufacturing process can comprise the following steps, as also illustrated in FIG.5.
  • Second, a direct production of the master microneedle array (134) from the CAD drawing can be produced by 3D direct laser writing using a non-dissolvable resin (e.g., IP-S photoresist) (step 122).
  • the 3D- ⁇ AM manufacturing system (136) can be, for example, a Nanoscribe 3D printing system.
  • a quick and high-fidelity replication of the master microneedle array 8123-111083-02 can be formed with a UV-curable resin (e.g., VeroWhitePlus, Tangoblack, Digital Materials) using a two-step micromolding approach (step 124).
  • This approach can include a negative elastomer mold (138) to form the replica (140) of the master microneedle array.
  • a microneedle array master mold can be created with multiple master microneedle array replicas (e.g., six replicas) on a 3D-printed microneedle array holder (step 126).
  • the 3D printed microneedle array holder (142) can be formed of, for example, a resin material.
  • microneedle array production molds (144) can be manufactured from the elastomer PDMS using micromolding (step 128).
  • microneedle arrays (150) can be fabricated (step 130) through a multiple-step spin-casting method using a centrifuge.
  • the microneedle arrays can have microneedles with or without undercut features incorporating one or more active components. An exemplary method for creating a microneedle array using the above outlined process is described below. Fabrication of master microneedle array.
  • a unique microneedle array design can be directly created from a 3D-CAD drawing using 3D laser printing (Nanoscribe Photonic Professional, GT) with the photopolymetric resist IP-S.
  • the Nanoscribe printing system was equipped with a laser generator, an optical cabinet, a Zeiss optical microscope attached to a lens to focus the laser beam, a Galvo mirror system to direct the laser-beam scanning, a piezoelectric stage for precise motion control, and an operation software (Nanowrite) to execute 3D printing. The whole system was placed on an optical table to eliminate vibrations during the printing process.
  • the microneedle array design was converted into ‘STL’ (StereoLithography) format.
  • the STL file was loaded into the specialized software (DeScribe, Germany) of the Nanoscribe system to select the processing conditions (i.e., the distance of slicing, hatching, and splitting). Finally, the STL file was converted into ‘GWL’ (General Writing Lithography) format to be exported in the Nanowrite software for printing the master microneedle array.
  • the master microneedle array was fabricated using Galvo-scan mode in XY plane and piezo-scan mode in Z direction.
  • the master microneedle array was split into blocks of 220 ⁇ m ⁇ 220 ⁇ m ⁇ 200 ⁇ m within the working range and then stitched together.
  • the laser power and writing speed was set to be 100 mW and 6 cm/s, respectively.
  • the master microneedle array was then printed through 8123-111083-02 two-photon polymerization of the IP-S photoresist by a femtosecond pulsed laser at a wavelength of 750 nm using a unique deep-in-liquid mode with the objective of 25 ⁇ and NA0.8 in Shell and Scaffold mode. After printing, the master microneedle array was developed in the photoresist solvent propylene glycol monomethyl ether acetate (PGMEA) for 30 min, followed by 5 min isopropyl alcohol (IPA) rinse.
  • PGMEA propylene glycol monomethyl ether acetate
  • IPA isopropyl alcohol
  • the master microneedle array was dried in the air, it was placed under a UV (365 nm) light with 16 mW/cm 2 intensity for 30 min to further crosslink the body to strengthen the microneedle array structure.
  • a two-stage micromolding method was performed. First, an elastomer mold which is negative of the master microneedle array was manufactured from polydimethylsiloxane (PDMS) using a micromolding approach. Elastomer molding using PDMS provides an accurate and reproducible replication of high-fidelity micron-scale structures.
  • PDMS polydimethylsiloxane
  • the master microneedle array was mounted in a petri-dish with a diameter of 5 cm and PDMS was obtained using the two-component curable silicone elastomer, SYLGARD® 184 (Dow Corning), by mixing the base material with a curing agent in 10:1 SYLGARD®-to-curing agent ratio. Subsequently, the mixture was poured over the master microneedle array mounted into the petri-dish and degassed for 15 min Next, the master microneedle array with the degassed mixture was placed in an oven and cured at 70° C. for 1 h. The cured PDMS was cooled down to room temperature for 5 min and then separated from the master microneedle array to obtain the negative PDMS mold.
  • SYLGARD® 184 Low Corning
  • the second processing step used the negative PDMS mold for fabrication of positive microneedle array replicas from the UV-curable resin (STRATASYS®, VeroWhiteplus- RGD835).
  • STRATASYS® VeroWhiteplus- RGD835
  • 20 ⁇ l of liquid (uncured) resin was poured onto the molds and then the molds were placed in a centrifuge to fill the microneedle-shaped wells with the resin at 4500 RPM and at 20° C. for 1 min.
  • the resin was then cured under UV light (365 nm) with 21.7 mW/cm 2 intensity for 5 min from each of the top and bottom sides to cure both the base and the microneedle tips.
  • microneedle array master molds are created through assembling six replicas of the master microneedle array onto the microneedle array holders fabricated by STRATASYS® from a non- dissolvable photo-polymer (VeroWhite) using a high-resolution (16 ⁇ m) Polyjet 3D printing system (Objet Connex 500 multi-material).
  • the 3D model of the microneedle array holders created using SolidWorks 2018 CAD software and then converted into the ‘STL’ (StereoLithography) file format.
  • the specialized software (Objet Studio) sliced this 3D model into 2D cross-sectional layers, creating a computer file that was sent to the 3D printer system.
  • the channels in the 3D printed microneedle array holder were designed and fabricated to serve as raised pockets in the microneedle array production molds to assist as reservoirs for both the active components and the base material of dissolvable microneedle arrays during the spin-casting process.
  • the created microneedle array master molds are baked at 80° C overnight in a vacuum oven to facilitate effective fabrication of elastomer microneedle array production molds.
  • Microneedle array production molds that include microneedle-shaped wells were fabricated from a commonly-used elastomer polydimethylsiloxane (PDMS) as described for the replication of the master microneedle array.
  • the base material was mixed with a curing agent in 10:1 SYLGARD®-to-curing agent ratio. Subsequently, the mixture was poured over the microneedle array master mold placed in a 10 cm diameter petri-dish and degassed for 15 min. Next, the master mold with the degassed mixture is placed in an oven to cure PDMS at 70° C for 1 h.
  • Microneedle array production molds such as those prepared by the above exemplary methods can then be used to fabricate polyphosphazene adjuvanted microneedle array vaccine through spin-casting (or spin-drying).
  • Cargo (including active components, and optionally non-active components) can be prepared at a desired useful concentration in a compatible solvent, such as aqueous polyphosphazene complexed vaccine antigens.
  • the solvents can be cargo specific, and can include a broad range of liquids such as water, organic polar and/or nonpolar liquids.
  • a base material can be used to form portions of each microneedle that have active components and portions that do not.
  • each microneedle can comprise active components only in the microneedle, or in some examples, 8123-111083-02 only in the upper half of the microneedle, or in other examples, only in a portion of the microneedle that tapers near the tip.
  • each microneedle preferably includes active components only in the portion that enters the skin.
  • the entire microneedle array can be made from polyphosphazene.
  • multi-stage spin- casting can be performed to integrate polyphosphazene adjuvanted vaccines in the tips of microneedles while forming the rest of the microneedles and the backing substrate from a water-soluble biomaterial, such as CMC.
  • CMC water-soluble biomaterial
  • various materials can be used as the base material. Described below are exemplary methods using CMC as the base material.
  • a CMC-based matrix can be formed at room temperature in a simple spin-casting and drying process, making CMC-microneedle arrays desirable for incorporation of sensitive biologics, peptides, proteins, nucleic acids, and other various active components.
  • CMC-hydrogel can be prepared from low viscosity sodium salt of CMC with or without active components in sterile dH2O.
  • CMC can be mixed with sterile distilled water (dH 2 O) and with the active components to achieve about 20 wt% CMC concentration.
  • the resulting mixture can be stirred to homogeneity and equilibrated at about 4 degrees Celsius for 24 hours.
  • the CMC and any other components can be hydrated and a hydrogel can be formed.
  • the hydrogel can be degassed in a vacuum for about an hour and centrifuged at about 20,000 g for an hour to remove residual micro-sized air bubbles that might interfere with a spin-casting/drying process of the CMC-microneedle arrays.
  • the dry matter content of the hydrogel can be tested by drying a fraction (10g) of it at 85 degrees Celsius for about 72 hours.
  • the ready-to-use CMC-hydrogel is desirably stored at about 4 degrees Celsius until use.
  • Active components can be incorporated in a hydrogel of CMC at a relatively high (20- 30%) CMC-dry active component weight ratio before the spin-casting process.
  • Arrays can be spin-cast at room temperature, making the process compatible with the functional stability of a structurally broad range of active components. Since the master and production molds can be reusable for a large number of fabrication cycles, the fabrication costs can be greatly reduced.
  • the resulting dehydrated CMC-microneedle arrays are generally stable at room temperature or slightly lower temperatures (such as about 4 degrees Celsius), and preserve the activity of the incorporated active components, facilitating easy, low-cost storage and distribution.
  • the surface of the production molds can be covered with about 50 ⁇ l (for molds with 11 mm diameter) of CMC-hydrogel and spin-cast by centrifugation at 2,500 g for about 5 minutes. After the initial CMC-hydrogel layer, another 50 ⁇ l CMC-hydrogel can be layered over the mold and centrifuged for about 4 hours at 2,500 g. At the end of a drying process, the CMC-microneedle arrays can be separated from the molds, trimmed off from excess material at the edges, collected and stored at about 4 degrees Celsius. The production molds can be cleaned and reused for further casting of microneedle arrays.
  • multiple loading cycles can be performed to achieve higher cargo loads as necessary for specific applications.
  • multiple cargos can be loaded in a single loading cycle as a complex solution, or as single solutions in multiple cycles (e.g., repeating the loading cycle described below) in accordance with specific cargo-compatibility requirements of individual cargos.
  • particulate cargos can be prepared as suspensions at the desired particle number/volume density.
  • a cargo’s working stock solution/suspension can be applied to the surface of microneedle array production molds at, for example, about 40 ⁇ l per cm 2 surface area.
  • the microneedle array production molds with cargo(s) can be centrifuged at 4500 rpm for 10 minutes to fill the microneedle array production molds needles with the working cargo stock.
  • the excess cargo solution/suspension can be removed, and the surface of the microneedle array production molds washed with 100 ⁇ l phosphate buffer saline (PBS) per cm 2 mold-surface area, or with the solvent used for the preparation of the cargo’s working stock.
  • PBS phosphate buffer saline
  • the microneedle array production molds containing the active cargo stock solution/suspension in the needle’s cavity can be spin-dried at 3500 rpm for 30 minutes at the required temperature with continues purging gas flow through the centrifuge at 0-50 L/min to facilitate concentration of the drying active cargo(s) in the needle-tips.
  • the purging gas can be introduced into the centrifuge chamber through tubular inlets.
  • Moisture content can be reduced using a dehumidifier tempered to the required temperature with recirculation into the centrifuge chamber.
  • the purging gas can be air, nitrogen, carbon dioxide or another inert or 8123-111083-02 active gas as required for specific cargo(s).
  • the flow rate is measured by flow-meters and controlled by a circulating pump device.
  • 100 ⁇ l 20% CMC90 hydrogel in H2O can be added to the surface microneedle array production molds’ per cm 2 microneedle array production mold area to load the structural component of the microneedle array.
  • the microneedle array production molds can be centrifuged at 4500 rpm for 10 min at the required temperature without purging gas exchange in the centrifuge chamber to fill up the microneedle array production molds needle cavities with the CMC90 hydrogel. This can be followed by a 30 min incubation period to enable rehydration of the active cargo(s) previously deposited in the microneedle array tips.
  • the microneedle array production molds can be centrifuged at 3500 rpm for 3 hours or longer at the required temperature with 0-50 L/min constant purging gas flow through the centrifuge chamber to spin-dry the microneedle array to less than 5% moisture content.
  • the dried microneedle array can then be separated from the microneedle array production molds for storage under the desired conditions.
  • CMC90 based microneedle array can be storable between about 50 0C to -86 0C.
  • Methods for Eliciting Immune Responses The disclosed microneedle arrays can be used to elicit an immune response.
  • the disclosed methods can also be used for treatment, such as for a tumor. In some aspects, these methods can be used to prevent infection by a pathogen, and/or prevent a disease caused by a pathogen.
  • the pathogen is a bacterium.
  • the pathogen is a virus.
  • the pathogen is a parasite or a fungus.
  • the microneedle arrays provided herein are for use in eliciting an immune response against a tumor, and/or preventing or reducing disease caused by the tumor, including, but not limited to, reducing the size and/or metastasis of the tumor.
  • methods of inducing an immune response against a tumor are provided.
  • the microneedle array includes a polyphosphazene adjuvant and one or more antigens from the tumor.
  • the use of the microneedle array induces a therapeutic immune response against a tumor cell in the subject, and wherein the therapeutic response is a reduction in tumor volume, tumor metastasis, or tumor number.
  • the tumor can be a hematological tumor.
  • hematological tumors include leukemias, including acute leukemias (such as 11q23-positive acute leukemia, acute lymphocytic 8123-111083-02 leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • acute leukemias such as 11q23-positive acute le
  • the tumor can be a solid tumor.
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma
  • Methods for eliciting an immune response against a pathogen in a subject, or protecting a subject from an infection or disease caused by the pathogen, comprise applying a microneedle array to an area of skin of the subject, thereby eliciting the immune response, or rendering the subject protected against the pathogen.
  • the microneedle array includes a polyphosphazene adjuvant and one or more antigens from the pathogen.
  • the microneedle array includes a polyphosphazene adjuvant and one or more antigens from a different pathogen, which is within the same genus as the pathogen against which the method is used to protect.
  • accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject.
  • These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such 8123-111083-02 risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and/or characterize an infection.
  • a microneedle array can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.
  • the microneedle arrays provided herein are for use in eliciting an immune response against the first pathogen, preventing infection by the first pathogen, and/or preventing a disease caused by the first pathogen.
  • a first antigen, and optionally a second antigen can be utilized from this first pathogen.
  • the first pathogen is a virus.
  • a method of eliciting an immune response against and/or protecting infection or a disease caused by a pathogen including applying a microneedle array including a polyphosphazene adjuvant and one or more antigens from the pathogen.
  • the pathogen includes a henipavirus, hepatitis C virus (HCV), filovirus, and influenza virus.
  • the microneedle arrays do not include an antigen from a second pathogen. In other aspects, the microneedle arrays include an antigen from a second pathogen.
  • the microneedle arrays provided herein are for use in eliciting an immune response against a second pathogen, preventing infection by the second pathogen, and/or preventing a disease caused by the second pathogen.
  • the second pathogen is from the same genus as the first pathogen.
  • the disclosed microneedle array by utilizing a polyphosphazene adjuvant in combination with one or more antigens from the first pathogen and utilizing immune cell-rich cutaneous microenvironments, generate potent immune responses, such as to the first and second pathogens, thereby providing cross-neutralization and cross-protection among different pathogens within the same genus.
  • the first and second pathogens are henipaviruses.
  • the first pathogen is a Hendra virus
  • the second pathogen is a Nipah virus.
  • the first pathogen is a Nipah virus
  • the second pathogen is a Hendra virus.
  • the first antigen is a G glycoprotein.
  • the disclosed microneedle arrays include cross-reactivity to a second pathogen without including an antigen from the second pathogen.
  • a method of eliciting an immune response against and/or protecting infection(s) or disease(s) caused by one or more pathogens within the same genus including applying a microneedle array including a polyphosphazene adjuvant, and an antigen from one pathogen of a genus.
  • the genus is Henipavirus.
  • the one pathogen is a Hendra virus or a Nipah virus.
  • the use of the microarray includes an immune response against a Nipah and/or Hendra virus.
  • the antigen included in the microarray is a G protein of a Hendra virus.
  • the immune response elicited upon application of a G protein polyphosphazene-based microneedle array generates potent antibody and cellular responses cross-reactive to multiple Henipavirus species, and thereby providing cross-protection against these species.
  • the method induces an immune response to a bacterium.
  • the polyphosphazene adjuvant is PCPP, fluorinated PCPP, PEGylated PCPP, poly[di(carboxylatophenoxy)phosphazene-co-di(trifluoroethoxy)phosphazene], or any combination thereof.
  • method includes an immune response to a virus.
  • the polyphosphazene adjuvant is PCEP, fluorinated PCEP, PEGylated PCEP, poly[di(carboxylatoethylphenoxy)phosphazene-co-di(trifluoroethoxy)phosphazene], or any combination thereof.
  • the microneedle array is pressed and held against an area of skin of a subject, such that the microneedles (or portions of the microneedles) will be inserted into the skin at a depth (e.g., reaching anywhere in the stratum comeum, penetrating the stratum comeum and reaching anywhere in the epidermis, or penetrating the epidermis and reaching anywhere in the dermis, dependent on the length of each microneedle) and will break off from the base portion.
  • the portions of the microneedles that enter the skin will dissolve in the skin fluid environment, releasing the active components.
  • One or more microneedle arrays can be provided to a subject, as a prime and boost, if desirous.
  • the immune response can include a humoral immune response, a cell-mediated immune response, or both.
  • an immune response induces myeloid cells.
  • an immune response induces IgM and/or IgG antibodies.
  • an immune response induces production of IFN ⁇ and/or IL-23.
  • a humoral response can be determined, for example, by a standard immunoassay for antibody levels in a serum sample from the subject receiving the microneedle array.
  • a cellular immune response is a response that typically involves T cells and can be determined in vitro or in vivo.
  • a general cellular immune response can be determined as the T cell proliferative 8123-111083-02 activity in cells (e.g., peripheral blood leukocytes (PBLs)) sampled from the subject at a suitable time following the administering of a microneedle array.
  • the percentage of proliferating T cells can be determined using flow cytometry.
  • Another way to measure cellular immunity involves measuring circulating frequencies of T cells secreting proinflammatory Type-1 and/or Type-17 cytokines in response to the antigen.
  • a suitable immunization regimen includes at least two separate inoculations with one or more microneedle arrays, with a second inoculation being administered more than about two, about three to eight, or about four, weeks following the first inoculation.
  • a third inoculation can be administered several months after the second inoculation, and in specific aspects, more than about five months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject’s “immune memory.”
  • the adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods.
  • the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of infection or progression to disease, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency to an uninfected partner. If such monitoring indicates that vaccination is sub- optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response.
  • a dose of one or more active components contained in the microneedle arrays can be increased or the route of administration can be changed.
  • the prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more, and can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses.
  • the immune response against the selected antigenic surface can be elicited by one or more inoculations of a subject.
  • Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in 8123-111083-02 the subject, or that elicit a desired response in the subject (such as a neutralizing immune response).
  • Suitable models in this regard include, for example, murine, rat, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art.
  • effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays).
  • an effective amount or effective dose of the composition may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes. Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery.
  • the actual dosage of disclosed active components will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.
  • a non-limiting range for an effective amount of an active component, such as an antigen, within the methods and immunogenic microneedle arrays of the disclosure is about 0.0001 mg/kg body weight to about 10 mg/kg body weight, such as about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, or about 10 mg/kg, for example, 0.01 mg/kg to about 1 mg/kg body weight, about 0.05 mg/kg
  • the dosage includes a set amount of a disclosed immunogen such as from about 1-300 ⁇ g, for example, a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 ⁇ g. 8123-111083-02
  • the dosage and number of doses will depend on the setting, for example, in an adult or anyone primed by prior infection or immunization, a single dose may be a sufficient booster. In na ⁇ ve subjects, in some examples, at least two doses would be given, for example, at least three doses. In some aspects, an annual boost is given, for example, along with an annual influenza vaccination. An infection does not need to be completely inhibited for the methods to be effective.
  • elicitation of an immune response to a pathogen can reduce or inhibit infection with the pathogen by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infected cells), as compared to infection in the absence of the therapeutic agent.
  • viral replication can be reduced or inhibited by the disclosed methods. Viral replication does not need to be completely eliminated for the method to be effective.
  • the immune response elicited using one or more of the disclosed immunogens can reduce viral replication by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable virus replication), as compared to viral replication in the absence of the immune response.
  • serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing.
  • Methods to assay for neutralization activity include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays (e.g., as described in Martin et al. (2003) Nature Biotechnology 21:71-76), and pseudovirus neutralization assays (e.g., as described in Georgiev et al. (Science, 340, 751-756, 2013), Seaman et al. (J. Virol., 84, 1439-1452, 2005), and Mascola et al. (J. Virol., 79, 10103-10107, 2005), each of which is incorporated by reference herein in its entirety.
  • PRNT plaque reduction neutralization
  • microneutralization assays e.g., microneutralization assays
  • flow cytometry based assays e.g., single-cycle infection assays (e.g., as described in Martin et al. (2003) Nature
  • Macrophage activity can also be assessed.
  • the expression of a cytokine, such as IL-23 or IFN- ⁇ can also be assessed.
  • administration of an effective amount of one or more of the disclosed immunogenic compositions to a subject elicits a neutralizing immune response in the subject.
  • Cellular immune responses can also be measured.
  • an mRNA is utlized.
  • dosage ranges of mRNA can be used in methods of the present disclosure. In one aspect, the dosage is in the range of about 0.1 to about 0.9 ⁇ g/day.
  • the dosage range can be about 0.1 to about 8123-111083-02 0.8 ⁇ g/day, about 0.1 to about 0.7 ⁇ g/day, about 0.1 to about 0.6 ⁇ g/day, about 0.1 to about 0.5 ⁇ g/day, about 0.1 to about 0.4 ⁇ g/day, about 0.1 to about 0.3 ⁇ g/day, or about 0.1 to about 0.2 ⁇ g/day.
  • the dosage range can be about 0.2 to about 0.9 ⁇ g/day, about 0.3 to about 0.9 ⁇ g/day, about 0.4 to about 0.9 ⁇ g/day, about 0.5 to about 0.9 ⁇ g/day, about 0.6 to about 0.9 ⁇ g/day, about 0.7 to about 0.9 ⁇ g/day, or about 0.8 to about 0.9 ⁇ g/day.
  • the dose can be about 0.1 ⁇ g/day, about 0.2 ⁇ g/day, about 0.3 ⁇ g/day, about 0.4 ⁇ g/day, about 0.5 ⁇ g/day, about 0.6 ⁇ g/day, about 0.7 ⁇ g/day, about 0.8 ⁇ g/day or about 0.9 ⁇ g/day.
  • the dosage is in the range of 1-10 ⁇ g/day. In another aspect, the dosage is 2-10 ⁇ g/day. In another aspect, the dosage is 3-10 ⁇ g/day. In another aspect, the dosage is 5-10 ⁇ g/day. In another aspect, the dosage is 2-20 ⁇ g/day. In another aspect, the dosage is 3- 20 ⁇ g/day. In another aspect, the dosage is 5-20 ⁇ g/day. In another aspect, the dosage is 10- 20 ⁇ g/day. In another aspect, the dosage is 3-40 ⁇ g/day. In another aspect, the dosage is 5-40 ⁇ g/day. In another aspect, the dosage is 10-40 ⁇ g/day. In another aspect, the dosage is 20-40 ⁇ g/day.
  • the dosage is 5-50 ⁇ g/day. In another aspect, the dosage is 10-50 ⁇ g/day. In another aspect, the dosage is 20-50 ⁇ g/day. In one aspect, the dosage is 1-100 ⁇ g/day. In another aspect, the dosage is 2-100 ⁇ g/day. In another aspect, the dosage is 3-100 ⁇ g/day. In another aspect, the dosage is 5-100 ⁇ g/day. In another aspect the dosage is 10-100 ⁇ g/day. In another aspect the dosage is 20-100 ⁇ g/day. In another aspect the dosage is 40- 100 ⁇ g/day. In another aspect the dosage is 60-100 ⁇ g/day. In another aspect, the dosage is 0.1 ⁇ g/day.
  • the dosage is 0.2 ⁇ g/day. In another aspect, the dosage is 0.3 ⁇ g/day. In another aspect, the dosage is 0.5 ⁇ g/day. In another aspect, the dosage is 1 ⁇ g/day. In another aspect, the dosage is 2 mg/day. In another aspect, the dosage is 3 ⁇ g/day. In another aspect, the dosage is 5 ⁇ g/day. In another aspect, the dosage is 10 ⁇ g/day. In another aspect, the dosage is 15 ⁇ g/day. In another aspect, the dosage is 20 ⁇ g/day. In another aspect, the dosage is 30 ⁇ g/day. In another aspect, the dosage is 40 ⁇ g/day. In another aspect, the dosage is 60 ⁇ g/day.
  • the dosage is 80 ⁇ g/day. In another aspect, the dosage is 100 ⁇ g/day. In another aspect, the dosage is 10 ⁇ g/dose. In another aspect, the dosage is 20 ⁇ g/dose. In another aspect, the dosage is 30 ⁇ g/dose. In another aspect, the dosage is 40 ⁇ g/dose. In another aspect, the dosage is 60 ⁇ g/dose. In another aspect, the dosage is 80 ⁇ g/dose. In another aspect, the dosage is 100 ⁇ g/dose. In another aspect, the dosage is 150 ⁇ g/dose. In another aspect, the dosage is 200 ⁇ g/dose. In another aspect, the dosage is 300 ⁇ g/dose. In another aspect, the dosage is 400 ⁇ g/dose.
  • the dosage is 600 ⁇ g/dose. In another aspect, the dosage is 800 ⁇ g/dose. In another aspect, the dosage is 1000 8123-111083-02 ⁇ g/dose. In another aspect, the dosage is 1.5 mg/dose. In another aspect, the dosage is 2 mg/dose. In another aspect, the dosage is 3 mg/dose. In another aspect, the dosage is 5 mg/dose. In another aspect, the dosage is 10 mg/dose. In another aspect, the dosage is 15 mg/dose. In another aspect, the dosage is 20 mg/dose. In another aspect, the dosage is 30 mg/dose. In another aspect, the dosage is 50 mg/dose. In another aspect, the dosage is 80 mg/dose. In another aspect, the dosage is 100 mg/dose.
  • the dosage is 10-20 ⁇ g/dose. In another aspect, the dosage is 20-30 ⁇ g/dose. In another aspect, the dosage is 20-40 ⁇ g/dose. In another aspect, the dosage is 30- 60 ⁇ g/dose. In another aspect, the dosage is 40-80 ⁇ g/dose. In another aspect, the dosage is 50-100 ⁇ g/dose. In another aspect, the dosage is 50-150 ⁇ g/dose. In another aspect, the dosage is 100-200 ⁇ g/dose. In another aspect, the dosage is 200-300 ⁇ g/dose. In another aspect, the dosage is 300-400 ⁇ g/dose. In another aspect, the dosage is 400-600 ⁇ g/dose. In another aspect, the dosage is 500-800 ⁇ g/dose.
  • the dosage is 800-1000 ⁇ g/dose. In another aspect, the dosage is 1000-1500 ⁇ g/dose. In another aspect, the dosage is 1500-2000 ⁇ g/dose. In another aspect, the dosage is 2-3 mg/dose. In another aspect, the dosage is 2-5 mg/dose. In another aspect, the dosage is 2-10 mg/dose. In another aspect, the dosage is 2-20 mg/dose. In another aspect, the dosage is 2-30 mg/dose. In another aspect, the dosage is 2-50 mg/dose. In another aspect, the dosage is 2-80 mg/dose. In another aspect, the dosage is 2-100 mg/dose. In another aspect, the dosage is 3-10 mg/dose. In another aspect, the dosage is 3-20 mg/dose. In another aspect, the dosage is 3-30 mg/dose.
  • the dosage is 3-50 mg/dose. In another aspect, the dosage is 3-80 mg/dose. In another aspect, the dosage is 3-100 mg/dose. In another aspect, the dosage is 5-10 mg/dose. In another aspect, the dosage is 5-20 mg/dose. In another aspect, the dosage is 5-30 mg/dose. In another aspect, the dosage is 5-50 mg/dose. In another aspect, the dosage is 5- 80 mg/dose. In another aspect, the dosage is 5-100 mg/dose. In another aspect, the dosage is 10-20 mg/dose. In another aspect, the dosage is 10-30 mg/dose. In another aspect, the dosage is 10-50 mg/dose. In another aspect, the dosage is 10-80 mg/dose. In another aspect, the dosage is 10-100 mg/dose.
  • FDA Food and Drug Administration
  • polyphosphazene and vaccine antigen into high- precision molds fabricated by 3D printing was used to create CMC/trehalose microneedle patches incorporating vaccine antigen with or without polyphosphazene adjuvant.
  • Evaluation of antigen-specific antibody responses Antigen-specific binding antibodies in serum were measured by indirect ELISAs. Serum was collected at the indicated time points. For each ELISA assay, Costar EIA/RIA plates (Corning Inc., Corning, NY) were coated with 100 ⁇ L/well of 1 ⁇ g/mL of corresponding antigen (e.g. HeV-sG) by overnight incubation at 4 °C.
  • corresponding antigen e.g. HeV-sG
  • Plates were washed three times with 0.05% Tween 20 in PBS and blocked with 1% normal goat serum (Jackson ImmunoResearch, West Grove, PA) in PBS for 1 h at 37 °C. Serum samples and standard antibodies were diluted in blocking buffer, added to plates, and incubated for 2 h at 37 °C. After washing (3x), plates were incubated for 1 h at 37 °C with biotinylated secondary antibodies (goat anti-mouse IgG, IgG1, or IgG2c; Jackson ImmunoResearch), diluted 1:20,000 in blocking buffer.
  • biotinylated secondary antibodies goat anti-mouse IgG, IgG1, or IgG2c; Jackson ImmunoResearch
  • T-cell responses to immunization were evaluated by intracellular cytokine staining and flow cytometry. Briefly, splenocytes were isolated from naive or immunized mice and RBCs lysed. Lymphocytes were isolated from lungs. Splenocytes (2 ⁇ 10 6 ) or lung resident 8123-111083-02 lymphocytes ( ⁇ 2-6 ⁇ 10 5 ) were stimulated in 96-well U-bottom plates (200 ⁇ L per well) with a pool of 12 HeV-sG peptides (Table 2).
  • Epitopes from the HeV sG antigen were identified using IEDB MHC-II Binding Predictions, MHC-I Binding Predictions, and MHC-I Processing Predictions (www.iedb.org); and corresponding peptides were synthesized by LifeTein (Somerset, NJ) (Table 2).
  • Peptides were dissolved in DMSO at 2 mg/mL and used at a final concentration of 5 ⁇ g/mL each in RPMI 1640 media (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; premium select, heat-inactivated; R&D Systems), 10 mM HEPES (Lonza, Walkersville, MD), 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Sigma), 1X antibiotic-antimycotic solution (Sigma), 1X non-essential amino acids (NEAA; Lonza), and 55 ⁇ M 2-mercaptoethanol (Gibco).
  • An Intracellular Fixation and Permeabilization Buffer Set (eBioscience) was used for intracellular cytokine staining, along with antibodies for CD154 (MR1, PE or PE-Cy7, BioLegend), IFN- ⁇ (XMG1.2, BV421, BD), TNF (MP6-XT22, Alexa 647, BD), IL-2 (JES6-5H4, Alexa 488, BD), and IL-4 (11B11, PE, BD). Surface and intracellular staining were performed in the presence of Brilliant Stain Buffer Plus (BD).
  • BD Brilliant Stain Buffer Plus
  • Multifunctional HeV-sG-specific T cells were identified by expression of activation induced marker CD154 (CD40L) and cytokine production, and quantified using the Boolean gating function in FlowJo. To account for non-specific activation, frequencies of CD154 + and/or cytokine positive cells from unstimulated controls were subtracted from corresponding peptide-stimulated samples, and negative values were set to zero.
  • rCedV Recombinant chimeric Cedar viruses
  • F fusion
  • G attachment
  • rCedV-NiV-B replication-competent chimeric viruses
  • G green fluorescent protein
  • rCedV-NiV-B-GFP or rCedV-HeV-GFP are then used in a quantitative fluorescence reduction neutralization test (FRNT).
  • Vero 76 cells were seeded at a density of 2 ⁇ 10 4 cells/well in black-walled clear bottom 96-well plates (Corning Life Sciences; Corning, NY, USA) and incubated for 24 h at 37 °C, 5% CO 2 . Serum is serially diluted 3- fold, starting with a dilution of 1:200, was used for a 7-point dose-response neutralization curve. An equal volume of DMEM-10 containing either rCedV-NiV-B-GFP or rCedV-HeV- GFP was added to each dilution for a final MOI of 0.05 and incubated for 2 h at 37 °C, 5% CO 2 .
  • Each of the virus-sera mixtures (90 ⁇ L/well) was added to the pre-seeded Vero 76 cells in triplicate and incubated for an additional 24 h at 37 °C, 5% CO2.
  • the virus-sera supernatants were removed, and the plates were fixed with 4% Formaldehyde in 1 ⁇ PBS for 20 min at room temperature. The plates were then washed 3 times by hand with a slow stream of diH 2 O, and the last wash was discarded before the plates were imaged using a CTL S6 analyzer (Cellular Technology Limited; Shaker Heights, OH, USA). Fluorescent foci were counted using the CTL Basic Count software.
  • ND 50 The 50% neutralization dose
  • the ND 50 values were calculated by non-linear regression curve fitting with a variable slope using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA).
  • the limit of detection for this assay was 50 fluorescent foci.
  • MNPs polyphosphazene adjuvanted dissolvable microneedle patches
  • FIGS.9A-9B show significantly Superior Immunogenicity of Polyphosphazene Adjuvanted MNP vaccines.
  • the vaccines generate potent, robust, and durable antigen-specific humoral and cellular immune responses, as shown in FIGS.10-13, 15, and 19-21.
  • FIGS.10A-10B show humoral responses elicited by polyphosphazene adjuvanted dissolvable MNP vaccines.
  • polyphosphazene adjuvanted MNP vaccines achieved induction of robust and long-lasting (72 weeks) virus-specific humoral responses (against HeV, FIGS.19A-19C) and cross-reactive humoral responses (against NiV, FIGS.19D-19E) in a prime-boost regime.
  • a single dose of PCEP adjuvanted MNP-based henipavirus vaccine also induced robust and long-lasting (52 weeks) virus-specific and cross- reactive humoral responses (FIGS.20A-20D).
  • HeV-sG + PCEP MNP vaccine is capable of inducing significantly higher binding and neutralizing antibody responses compared to HeV-sG MNP, which are robust, long-lasting, and cross-reactive, demonstrating the potent and durable humoral immunogenicity characteristics of polyphosphazene adjuvanted MNP vaccines. 8123-111083-02 Further studies were performed to study the immunogenicity of MNP vaccines in eliciting cellular immune responses.
  • Polyphosphazene adjuvanted MNP vaccines e.g., HeV- sG + PCEP
  • elicit potent and strong systemic polyfunctional T-cell responses as demonstrated by significant higher levels of antigen-specific CD4 + and CD8 + T cells in mice immunized with polyphosphazene adjuvanted MNP vaccines (FIGS.15A-15B).
  • cellular responses elicited by PCEP or PCPP adjuvanted, HeV-sG MNP vaccines were measured.
  • HeV-sG + PCEP MNP or HeV-sG + PCPP MNP vaccine were compared to cellular responses induced by intramuscularly injected Alhydrogel-complexed HeV-sG (HeV-sG + Alum I.M.: the gold standard of HeV-sG vaccine) (FIGS.21A-21C).
  • Cellular immune responses induced by HeV-sG + PCEP MNP were superior to those elicited by HeV-sG + Alum I.M. and HeV-sG + PCPP MNP.
  • polyphosphazene adjuvanted MNP vaccines demonstrated superior immunogenicity compared to traditional intramuscular injection of conventional adjuvant complexed antigens, for antigens from a broad spectrum of pathogens including SARS-CoV- 2 virus, influenza virus, Sudan ebolavirus, and Hepatitis C virus (FIGS.17A-17D).
  • FIG.17A-17D Polyphosphazene adjuvanted MNP vaccines carrying different antigens from different viruses invariably exhibit superior immunogenicity.
  • Figure 17A shows higher serum concentrations of SARS-CoV-2 spike protein (SP)- specific total IgG in mice immunized by polyphosphazene adjuvanted MNP vaccine, compared to those in mice immunized by traditional intramuscular injection of conventional aluminum adjuvanted SARS-CoV-2 SP vaccine.
  • Figure 17B shows compatibility and antigen-specific immune-enhancing characteristics of PCEP with influenza HA antigen in dissolvable microneedle patch format.
  • SP SARS-CoV-2 spike protein
  • Figure 17C shows higher serum levels of Sudan virus GP-specific total IgG in mice immunized by polyphosphazene adjuvanted MNP vaccine, compared to those in mice immunized by traditional intramuscular injection of conventional aluminum adjuvanted Sudan virus GP vaccine.
  • Figure 17D shows higher serum levels of Hepatitis C virus sE1E2-specific total IgG in mice immunized by polyphosphazene adjuvanted MNP vaccine, compared to those in mice immunized by traditional intramuscular injection of conventional aluminum adjuvanted Hepatitis C virus sE1E2 vaccine.
  • Example 6 Combining Multiple Adjuvants in MNP Vaccine Combining polyphosphazene with certain adjuvants in an MNP-based vaccine can further enhance antigen-specific protective immune responses.
  • Combining the TLR3 agonist 8123-111083-02 poly(I:C) with PCEP in an MNP with HeV sG significantly enhanced humoral and cellular immune responses against HeV sG, particularly the level of antigen-specific antibodies and antigen-specific CD4 + cells generated.
  • Figure 18A shows antigen-specific antibody responses.
  • Figures 18B-18C show antigen-specific cellular immune responses.
  • Example 7 Feasibility of Polyphosphazene Adjuvanted MNP Vaccine for Human Skin Immunization
  • Living human skin explant models established with freshly excised human skin represent a useful platform for validating the feasibility of skin-targeted vaccines for human immunization.
  • a polyphosphazene adjuvanted MNP-based HNV vaccine was engineered by incorporating the immunogen HeV-sG with the polyphosphazene adjuvant PCEP into MNPs and studies were performed using living human skin explants. MNPs were applied to human skin explants for 5 minutes, and the explants were cultured for 48 hours.
  • HeV-sG + PCEP MNP enhanced the frequencies of migrating Langerhans cells (LCs) and CD1a + dermal dendritic cells (DCs) and reduced the frequence of migrating CD14 + dermal DCs as compared to HeV-sG MNP (FIG.23A).
  • LCs migrating Langerhans cells
  • DCs dermal dendritic cells
  • HLA-DR + APCs migrating from human skin after HeV-sG + PCEP MNP application expressed significantly higher levels of co-stimulatory molecules and CCR7 compared to those from HeV-sG MNP treated skin (FIG.23B), consistent with more immunostimulatory and migratory APC phenotypes that are associated with improved adaptive immune responses.
  • LNs skin draining lymph nodes
  • polyphosphazene adjuvanted MNP vaccines enable the effective modulation of the human skin microenvironment to induce potent APCs with immunostimulatory phenotypes. 8123-111083-02
  • the polyphosphazene adjuvanted MNP vaccines are safe, thermostable, and needle- free vaccines with simplified delivery, storage, distribution, and disposal, and improved acceptability compared to conventional needle and syringe vaccines.
  • a polyphosphazene adjuvanted MNP-based HNV vaccine was engineered by incorporating the immunogen HeV-sG with PCEP into MNPs, and its safety and temperature stability were tested in mice.
  • HeV-sG + PCEP MNPs have good safety profiles, since there were no significant changes in animal weight (FIG.24B), body temperature (FIG. 24C), and serum levels of IL-6 cytokine (FIG.24D), which is an established biomarker of systemic toxicity and fever, before and after the application of MNPs.
  • polyphosphazene adjuvanted MNP vaccines retain their immunogenicity after an extended period of storage, even when exposed to environmental stress (FIGS.14 and 25A-25B).
  • Polyphosphazene adjuvanted MNP vaccines are stable under room temperature, obviating the need for refrigeration, as demonstrated by their retained immunogenicity for at least 1 month storage at room temperature (FIG.14).
  • HeV-sG + PCEP MNP vaccines retained their immunogenicity at room temperature (RT, 22 °C) for at least a year, indicated by the non-significant difference in antigen-specific total IgG antibody levels induced by freshly prepared HeV-sG + PCEP MNP vaccines and HeV-sG + PCEP MNP vaccines that had been stored in a sealed vial at RT for a year (FIGS 25A-25B).
  • HeV-sG + PCEP MNP vaccines that were exposed to gamma irradiation at a dose of 25 kilogray (the irradiation dose recommended by regulatory agencies to render medical MNPs sterile) and then stored at RT in a sealed vial for a year also retained their immunogenicity (FIGS 25A-25B).
  • HeV-sG + PCEP MNP vaccines Consistent with ambient temperature of current HNV endemic-prone regions, the immunogenicity of HeV-sG + PCEP MNP vaccines was preserved even at an elevated temperature, 40 °C, for at least 3 months, as revealed by the comparable antigen-specific total IgG antibody levels generated by fresh HeV-sG + PCEP MNP vaccines and HeV-sG + PCEP MNP vaccines that had been stored in a sealed vial at 40 °C for 3 months.
  • polyphosphazene adjuvanted MNP vaccines offer favorable globally deployable vaccine product characteristics, including safety for well tolerability, thermostability for cost- 8123-111083-02 effective storage and widespread distribution (without the need for costly and complex cold chain distribution networks), and the feasibility for terminal sterilization via gamma irradiation.

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Abstract

L'invention concerne des vaccins thermostables, largement applicables, sans aiguille, ciblés sur la peau, à base de timbre à micro-aiguilles solubles et à adjuvant de polyphosphazène.
PCT/US2025/019036 2024-03-08 2025-03-07 Vaccins à matrice de micro-aiguilles à adjuvant de polyphosphazène Pending WO2025189158A1 (fr)

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